Cdna for human methylenetetrahydrofolate reductase and uses thereof

ABSTRACT

The invention features methods for diagnosing subjects at risk for or suffering from a disease or disorder, such as a psychosis. Methods are also provided for selecting a preferred therapy for a particular subject or group of subjects.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.11/067,779, filed Feb. 28, 2005, which is a continuation of U.S. patentapplication Ser. No. 10/235,172, filed Sep. 4, 2002, which is acontinuation of U.S. patent application Ser. No. 09/592,595, filed Jun.12, 2000, which is a continuation-in-part of U.S. patent applicationSer. No. 09/258,928 filed Mar. 1, 1999, which is a continuation in partof U.S. patent application Ser. No. 08/738,000 filed Feb. 12, 1997,which is the National Stage of international application numberPCT/CA95/00314, filed May 25, 1995, which claims priority to UnitedKingdom patent application number 9410620.0, filed May 26, 1994, each ofwhich is hereby incorporated by reference.

BACKGROUND OF THE INVENTION

(a) Field of the Invention

The invention relates to a cDNA probe for humanmethylenetetrahydrofolate reductase (MTHFR), and its uses.

(b) Description of Prior Art

Folic acid derivatives are coenzymes for several critical single-carbontransfer reactions, including reactions in the biosynthesis of purines,thymidylate and methionine. Methylenetetrahydrofolate reductase (MTHFR;EC 1.5.1.20) catalyzes the NADPH-linked reduction of5,10-methylenetetrahydrofolate to 5-methyltetrahydrofolate, aco-substrate for methylation of homocysteine to methionine. The porcineliver enzyme, a flavoprotein, has been purified to homogeneity; it is ahomodimer of 77-kDa subunits. Partial proteolysis of the porcine peptidehas revealed two spatially distinct domains: an N-terminal domain of 40kDa and a C-terminal domain of 37 kDa. The latter domain contains thebinding site for the allosteric regulator S-adenosylmethionine.

Hereditary deficiency of MTHFR, an autosomal recessive disorder, is themost common inborn error of folic acid metabolism. A block in theproduction of methyltetrahydrofolate leads to elevated homocysteine withlow to normal levels of methionine. Patients with severe deficiencies ofMTHFR (0-20% activity in fibroblasts) can have variable phenotypes.Developmental delay, mental retardation, motor and gait abnormalities,peripheral neuropathy, seizures and psychiatric disturbances have beenreported in this group, although at least one patient with severe MTHFRdeficiency was asymptomatic. Pathologic changes in the severe forminclude the vascular changes that have been found in other conditionswith elevated homocysteine, as well as reduced neurotransmitter andmethionine levels in the CNS. A milder deficiency of MTHFR (35-50%activity) has been described in patients with coronary artery disease(see below). Genetic heterogeneity is likely, considering the diverseclinical features, the variable levels of enzyme activity, and thedifferential heat inactivation profiles of the reductase in patients'cells.

Coronary artery disease (CAD) accounts for 25% of deaths of Canadians.Cardiovascular risk factors (male sex, family history, smoking,hypertension, dyslipoproteinemia and diabetes) account for approximately60 to 70% of the ability to discriminate CAD patients from healthysubjects. Elevated plasma homocysteine has also been shown to be anindependent risk factor for cardiovascular disease.

Homocysteine is a sulfhydryl-containing amino acid that is formed by thedemethylation of methionine. It is normally metabolized to cysteine(transsulfuration) or re-methylated to methionine. Inborn errors ofmetabolism (as in severe MTHFR deficiency) causing extreme elevations ofhomocysteine in plasma, with homocystinuria, are associated withpremature vascular disease and widespread arterial and venous thromboticphenomena. Milder elevations of plasma homocysteine (as in mild MTHFRdeficiency) have been associated with the development of peripheralvascular disease, cerebrovascular disease and premature CAD.

Homocysteine remethylation to methionine requires the folic acidintermediate, 5-methyltetrahydrofolate, which is produced from5,10-methylenetetrahydrofolate folate through the action of5,10-methylenetetrahydrofolate reductase (MTHFR). Deficiency of MTHFRresults in an inability to metabolize homocysteine to methionine;elevated plasma homocysteine and decreased methionine are the metabolicconsequences of the block. Severe deficiencies of MTHFR (less than 20%of activity of controls) as described above, are associated withearly-onset neurologic symptoms (mental retardation, peripheralneuropathy, seizures, etc.) and with atherosclerotic changes andthromboembolism. Milder deficiencies of MTHFR (35-50% of activity ofcontrols), with a thermolabile form of the enzyme, are seen in patientswith cardiovascular disease without obvious neurologic abnormalities.

In a survey of 212 patients with proven coronary artery disease, thethermolabile form of MTHFR was found in 17% of the CAD group and 5% ofcontrols. In a subsequent report on 339 subjects who underwent coronaryangiography, a correlation was found between thermolabile MTHFR and thedegree of coronary artery stenosis. Again, traditional risk factors(age, sex, smoking, hypertension, etc.) were not significantlyassociated with thermolabile MTHFR. All the studies on MTHFR wereperformed by enzymatic assays of MTHFR in lymphocytes, with measurementsof activity before and after heat treatment to determine thermolabilityof the enzyme.

Since 5-methyltetrahydrofolate, the product of the MTHFR reaction, isthe primary form of circulatory folate, a deficiency in MTHFR might leadto other types of disorders. For example, periconceptual folateadministration to women reduces the occurrence and recurrence of neuraltube defects in their offspring. Neural tube defects are a group ofdevelopmental malformations (meningomyelocele, anencephaly, andencephalocele) that arise due to failure of closure of the neural tube.Elevated levels of plasma homocysteine have been reported in mothers ofchildren with neural tube defects. The elevated plasma homocysteinecould be due to a deficiency of MTHFR, as described above forcardiovascular disease.

Neuroblastomas are tumors derived from neural crest cells. Many of thesetumors have been reported to have deletions of human chromosome region1p36, the region of the genome to which MTHFR has been mapped. It ispossible that MTHFR deletions/mutations are responsible for orcontribute to the formation of this type of tumor. MTHFR abnormalitiesmay also contribution to the formation of other types of tumors, such ascolorectal tumors, since high dietary folate has been shown to beinversely associated with risk of colorectal carcinomas.

MTHFR activity is required for homocysteine methylation to methionine.Methionine is necessary for the formation of S-adenosylmethionine, theprimary methyl donor for methylation of DNA, proteins, lipids,neurotransmitters, etc. Abnormalities in MTHFR might lead to lowerlevels of methionine and S-adenosylmethionine, as well as to elevatedhomocysteine. Disruption of methylation processes could result in a widevariety of conditions, such as neoplasias, developmental anomalies,neurologic disorders, etc.

Although the MTHFR gene in Escherichia coli (metF) has been isolated andsequenced, molecular studies of the enzyme in higher organisms have beenlimited without the availability of an eukaryotic cDNA.

It would be highly desirable to be provided with a cDNA probe for humanmethylenetetrahydrofolate reductase (MTHFR). This probe would be usedfor identification of sequence abnormalities in individuals with severeor mild MTHFR deficiency, including cardiovascular patients and patientswith neurologic symptoms or tumors. The probe would also be used in genetherapy, isolation of the gene, and expression studies to produce theMTHFR protein. The probe would also provide the amino acid sequence ofthe human MTHFR protein, which would be useful for therapy of MTHFRdeficiency by biochemical or pharmacological approaches.

It would be highly desirable to be provided with a molecular descriptionof mutations in methylenetetrahydrofolate reductase deficiency.

Patients with sequence abnormalities in MTHFR might have differentresponses to drugs, possibly but not limited to drugs that affect folatemetabolism. Therefore, it would be useful to know if these mutations arepresent before determining the appropriate therapy. The drugs/diseasesfor which this might be relevant include cancer chemotherapeutic agents,antibiotics, antiepileptic medication, antiarthritic medication, etc.

SUMMARY OF THE INVENTION

One aim of the present invention is to provide a cDNA probe for humanmethylenetetrahydrofolate reductase (MTHFR).

Another aim of the present invention is to provide a moleculardescription of mutations in methylenetetrahydrofolate reductasedeficiency.

Another aim of the present invention is to provide a nucleic acid andamino acid sequence for human methylenetetrahydrofolate reductase.

Another aim of the present invention is to provide potential therapy forindividuals-with methylenetetrahydrofolate reductase deficiency.

Another aim of the present invention is to provide a system forsynthesis of MTHFR protein in vitro.

A further aim of the present invention is to provide technology/protocolfor identification of sequence changes in the MTHFR gene.

In accordance with one aspect of the present invention, there isprovided a cDNA probe for human methylenetetrahydrofolate reductase(MTHFR) gene encoded by a nucleotide sequence as set forth in SEQ IDNO:1 or having an amino acid sequence as set forth in SEQ ID NO:2. Theprobe comprises a nucleotide sequence that hybridizes to the MTHFRnucleotide sequence, or an amino acid sequence that hybridizes to theMTHFR amino acid sequence.

In accordance with another aspect of the present invention, there isprovided a method of diagnosis of methylenetetrahydrofolate reductase(MTHFR) deficiency in a patient with MTHFR deficiency. The methodcomprises the steps of amplifying a DNA sample obtained from the patientor reverse-transcripting a RNA sample obtained from the patient into aDNA and amplifying the DNA, and analyzing the amplified DNA to determineat least one sequence abnormality with respect to a human MTHFR encodedby a nucleotide sequence as set forth in SEQ ID NO:1 or having an aminoacid sequence as set forth in SEQ ID NO:2, the sequence abnormalitybeing indicative of MTHFR deficiency.

The sequence abnormality may comprise a mutation selected from a groupconsisting of 167G/A, 482G/A, 559C/T, 677C/T, 692C/T, 764C/T, 792+1G/A,985C/T, 1015C/T, 1081C/T, 1298A/C and 1317T/C.

The selected mutation may consist of 677C/T.

The MTHFR deficiency may be associated with a disease, disorder, ordysfunction selected from a group consisting of cardiovasculardisorders, cancer, osteoporosis, increased risk of occurrence of aneural tube defect in an offspring of said patient, neurologicaldisorders, disorders influenced by folic acid metabolism, metabolic orendocrine disease, inborn errors of metabolism, inflammation, immunedisorders, psychiatric illness, neoplastic disease and other relateddisorders, and renal disease.

The cancer may be selected from a group consisting of neuroblastomas andcolorectal carcinomas.

The disorder may consist of osteoporosis.

In accordance with yet another aspect of the present invention, there isprovided a method for gene therapy of methylenetetrahydrofolatereductase (MTHFR) deficiency in a patient. The method comprises thesteps of producing a recombinant vector for expression of MTHFR underthe control of a suitable promoter, the MTHFR being encoded by anucleotide sequence as set forth in SEQ ID NO:1 or having an amino acidsequence as set forth in SEQ ID NO:2, and transfecting the patient withthe vector for expression of MTHFR.

In accordance with yet another aspect of the present invention, there isprovided a human methylenetetrahydrofolate reductase (MTHFR) proteinencoded by a nucleotide sequence as set forth in SEQ ID NO:1 or havingan amino acid sequence as set forth in SEQ ID NO:2.

In accordance with yet another aspect of the present invention, there isprovided a recombinant human methylenetetrahydrofolate reductase (MTHFR)protein encoded by a nucleotide sequence as set forth in SEQ ID NO:1 orhaving an amino acid sequence as set forth in SEQ ID NO:2.

In accordance with yet another aspect of the present invention, there isprovided a method of treatment of MTHFR-deficiency in a patient thatcomprises administering such a MTHFR protein.

The MTHFR deficiency may be associated with a cancer.

The cancer may be selected from a group consisting of neuroblastomas andcolorectal carcinomas.

In accordance with yet another aspect of the present invention, there isprovided a method of preventing an occurrence of a neural tube defect inan offspring of a patient. The method comprises administering to thepatient such a MTHFR protein.

In accordance with yet another aspect of the present invention, there isprovided a method for determining drug susceptibility, drug response ordrug toxicity of a patient having a methylenetetrahydrofolate reductase(MTHFR) deficiency. The method comprises the steps of amplifying a DNAsample obtained from the patient or reverse-transcripting a RNA sampleobtained from the patient into a DNA and amplifying said DNA, analyzingthe amplified DNA to determine a sequence abnormality in a MTHFRsequence, the MTHFR sequence being encoded by a nucleotide sequence asset forth in SEQ ID NO:1 or having an amino acid sequence as set forthin SEQ ID NO:2, and administering the drug to the patient anddetermining the sequence abnormality associated with the patientsusceptibility, response or toxicity to the drug. Further, in humantherapeutic approaches, both pharmacokinetics and pharmacodynamics maybe influenced by folic acid metabolism. Pharmacokinetics includes, butis not excluded to, factors such as absorption, distribution,metabolism, and excretion. Other considerations include safety andtoxicity. Examples of toxicities from therapeutics include, but are notlimited to, blood dyscrasias, cutaneous toxicities, systemic toxicity,CNS toxicity, hepatic toxicity, cardiovascular toxicity, pulmonarytoxicity, and renal toxicity.

The sequence abnormality may comprise a mutation selected from a groupconsisting of 167G/A, 482G/A, 559C/T, 677C/T, 692C/T, 764C/T, 792+1G/A,985C/T, 1015C/T, 1081C/T, 1298A/C and 1317T/C and the drug may beselected from a group consisting of cancer chemotherapeutic agents,antibiotics, antiepileptic agents, antiarthritic agents, andanti-inflammatory agents. Examples of chemotherapeutic agents includealkylating agents (nitrogen mustards, ethylenimines and methylmelamines,alkyl sulfonates, nitrosoureas, triazenes), antimetabolites (folic acidanalogs, pyrimidine analogs, purine analogs and related inhibitors), andnatural products (vinca alkyloids, epipodophyllotoxins, antibiotics,enzymes, biological response modifiers). Examples of anti-inflammatoryagents include non-steroidal anti-inflammatory agents, steroids,antihistaminergics, 5-LO inhibitors, cytokine agonists, and cytokineantagonists. The drug may also include therapies for metabolic orendocrine disease such as hormone agonists and antagonists,intracellular response modifiers, and secretegogues. Other possibledrugs include therapies for cardiovasular disease or disorders such asantianemic, antiangina, antiarrythmics, antihypertensives, positiveinotropic agents, and antithrombotics. The drug may also be a therapyfor an immune disorder, such as immune modulators, immunosuppressionagents, and immunostimulation agents. Additionally, any other drug forthe treatment of a metabolic disease, inborn errors of metabolism,psychiatric disorders, neoplastic disease and other related disorders,and renal disease is relevant to the invention

The MTHFR deficiency may be associated with a disorder selected from agroup consisting of cardiovascular disorders, coronary and arterialdisorders, neurological disorders, increased risk of occurrence of aneural tube defect in an offspring, cancer, osteoporosis and otherdisorders influenced by folic acid metabolism.

In accordance with yet another aspect of the present invention, there isprovided a method of treatment of a patient having a cancer comprisingthe step of inhibiting gene expression for a MTHFR protein or a mRNAproduced form the gene.

In accordance with yet another aspect of the present invention, there isprovided a method of treatment of a patient having a cancer comprisingthe step of inhibiting the MTHFR protein.

The use of MTHFR alleles as diagnostic, therapeutic, prognostic, andpharmacogenomic markers may also be applied to neurological disorderssuch as psychoses and to other diseases and disorders that may betreated with anti-psychotic therapeutics. The presence of particularMTHFR alleles may be used to predict the safety and efficacy of aparticular therapy and to select preferred therapies for these subjects.

Accordingly, in one aspect, the invention provides a method ofdiagnosing a psychosis in a subject. This method involves analyzing theMTHFR nucleic acid in a sample obtained from the subject and determiningthe presence of at least one heterozygous MTHFR mutant allele in thesubject that is indicative of the subject having the psychosis.

In a related aspect, the invention features a method of determining arisk for a psychosis or a propensity for a psychosis in a subject. Thismethod includes analyzing the MTHFR nucleic acid in a sample obtainedfrom said subject and determining the presence of at least oneheterozygous MTHFR mutant allele in the subject that is indicative ofthe risk for a psychosis or the propensity for a psychosis in thesubject.

In another aspect, the invention provides a method of diagnosing apsychosis in a subject. This method involves analyzing the MTHFR nucleicacid in a sample obtained from the subject and determining the presenceof a heterozygous MTHFR mutation at position 677 and/or the presence ofat least one other MTHFR mutation at a position other than 677. Thesemutations are indicative of the subject having the psychosis.

The invention also provides a related method of diagnosing a risk for apsychosis or a propensity for a psychosis in a subject. This methodincludes analyzing the MTHFR nucleic acid in a sample obtained from thesubject and determining the presence of a heterozygous MTHFR mutation atposition 677 and the presence of at least one other MTHFR mutation at aposition other than 677. These mutations are indicative of the risk fora psychosis or the propensity for a psychosis in the subject.

In addition to their use in diagnosing a psychosis or an increased riskfor a psychosis, the determination of one or more mutations in an MTHFRallele may also be used to classify subjects into a subgroup for aclinical trial of an anti-psychotic therapy, to determine a preferredanti-psychotic therapy for a subject, or to determine whether the MTHFRmutations are indicative of a response to an anti-psychotic therapy.

Accordingly, the invention also provides a method for stratification ofsubjects involved in a clinical trial of an anti-psychotic therapy. Thismethod involves analyzing the MTHFR nucleic acid in a sample obtainedfrom a subject and determining before, during, or after the clinicaltrial the presence of at least one MTHFR mutant allele in the subjectthat places the subject into a subgroup for the clinical trial. In onepreferred embodiment, the anti-psychotic therapy is a therapy forschizophrenia.

In another aspect, the invention provides a method for selecting atherapy for a subject suffering from a psychosis. This method includeanalyzing the MTHFR nucleic acid in a sample obtained from the subjectand determining the presence of at least one MTHFR mutant allele in thesubject that is indicative of the safety or efficacy of the therapy.

In yet another aspect, the invention features a method for determiningwhether a mutant MTHFR allele is indicative of a response to a therapyfor a psychosis. This method includes (a) determining whether theresponse of a first subject or set of subjects at increased risk for ordiagnosed with the psychosis differs from the response of a secondsubject or set of subjects at increased risk for or diagnosed with thepsychosis, (b) analyzing the MTHFR nucleic acid in a sample obtainedfrom the first and second subjects or sets of subjects, and (c)determining whether at least one MTHFR mutant allele differs between thefirst and second subjects or sets of subjects. If the MTHFR mutantallele is correlated to a response to the therapy, the mutant allele isdetermined to be indicative of the safety or efficacy of the therapy.

The invention also provides a method for preventing, delaying, ortreating a psychosis in a subject. This method includes (a) analyzingthe MTHFR nucleic acid in a sample obtained from the subject, (b)determining the presence of at least one MTHFR mutant allele in thesubject that is predictive of the safety or efficacy of at least oneanti-psychotic therapy, (c) determining a preferred therapy for thesubject, and (d) administering the preferred therapy to the subject.

In another aspect, the invention provides a pharmaceutical compositionincluding a compound which has a differential effect in subjects havingat least one copy of a particular MTHFR allele and has apharmaceutically acceptable carrier, excipient, or diluent. Thiscompound is preferentially effective to treat a subject having theparticular MTHFR allele. In a preferred embodiment, the composition isadapted to be preferentially effective based on the unit dosage,presence of additional active components, complexing of the compoundwith stabilizing components, or inclusion of components enhancingdelivery or slowing excretion of the compound. In another preferredembodiment, the compound is deleterious to subjects having at least onecopy of the particular MTHFR allele or in subjects not having at leastone copy of the particular MTHFR allele, but not in both. In yet anotherpreferred embodiment, the subject suffers from a disease or conditionselected from the group consisting of amyotrophic lateral sclerosis,anxiety, dementia, depression, epilepsy, Huntington's disease, migraine,demyelinating disease, multiple sclerosis, pain, Parkinson's disease,schizophrenia, psychoses, and stroke. In still another preferredembodiment, the pharmaceutical composition is subject to a regulatoryrestriction or recommendation for use of a diagnostic test determiningthe presence or absence of at least one particular MTHFR allele. Inanother preferred embodiment, the pharmaceutical composition is subjectto a regulatory limitation or recommendation restricting or recommendingrestriction of the use of the pharmaceutical composition to subjectshaving at least one particular MTHFR allele. In yet another preferredembodiment, the pharmaceutical composition is subject to a regulatorylimitation or recommendation indicating the pharmaceutical compositionis not to be used in subjects having at least one particular MTHFRallele. In still another preferred embodiment, the pharmaceuticalcomposition is packaged, and the packaging includes a label or insertrestricting or recommending the restriction of the use of thepharmaceutical composition to subjects having at least one particularMTHFR allele. In yet another embodiment, the pharmaceutical compositionis packaged, and the packaging includes a label or insert requiring orrecommending the use of a test to determine the presence or absence ofat least one particular MTHFR allele in a subject. Preferably, thecompound is an anti-psychotic therapy, such as a therapy forschizophrenia.

In preferred embodiments of each of the aspects of the invention, themutant MTHFR allele encodes an MTHFR protein with reduced activity orreduced thermal stability. It is also contemplated that the MTHFRprotein may have a reduced half-life. Preferably, the MTHFR mutation isa missense mutation. Preferred MTHFR mutations include a G/A mutation atposition 167, a G/A mutation at position 482, a C/T mutation a position559, a C/T mutation at position 677, a C/T mutation at position 692, aC/T mutation at position 764, a G/A mutation at position 792+1, a C/Tmutation at position 985, a C/T mutation at position 1015, a C/Tmutation at position 1081, an A/C mutation at position 1298, and a T/Cmutation at position 1317. It is also contemplated that the mutations atthese positions may involve changes to other nucleotides. In otherpreferred embodiments, the subject has at least two MTHFR mutantalleles. Preferably the two or more mutant MTHFR alleles have at leastone of the following: a G/A mutation at position 167, a G/A mutation atposition 482, a C/T mutation a position 559, a C/T mutation at position677, a C/T mutation at position 692, a C/T mutation at position 764, aG/A mutation at position 792+1, a C/T mutation at position 985, a C/Tmutation at position 1015, a C/T mutation at position 1081, an A/Cmutation at position 1298, or a T/C mutation at position 1317. In yetother preferred embodiments, the MTHFR mutation results in a change inthe amino acid at position 226 of the encoded protein from alanine foundin wild-type MTHFR to any other amino acid, including valine or residuesother than valine. In still other preferred embodiments, the mutationresults in a change in amino acid 433 of the encoded protein fromglutamic acid to any other amino acid, including alanine or residuesother than alanine. When two mutations are present at differentnucleotide positions, the mutations may be in the same or differentMTHFR alleles. The MTHFR mutation may, without limitation, also be aninsertion, deletion, or frameshift mutation.

Preferably, the psychosis being diagnosed or treated is schizophrenia.The psychosis may also be any other psychosis such as manic-depressivedisease, depression with psychotic features, organic psychoticdisorders, psychosis in alcohol or drug intoxication, postinfectionpsychosis, postpartum psychosis, senile psychosis, traumatic psychosis,manic-depressive psychosis, psychosis from toxic agents, and acuteidiopathic psychotic illnesses.

The use of MTHFR alleles as diagnostic, therapeutic, prognostic, andpharmacogenomic markers may also be applied to other neurologicaldisorders. Examples of neurological disorders include, but are notlimited to, mood disorders, neurodegenerative diseases, cognitivedisorders, seizure disorders such as epilepsy, headaches such asmigraines, pain associated with the central nervous system,demyelinating diseases, spasticity, neural tube defects,neurofibromatosis, and ischemic cerebrovascular diseases such asthrombotic and hemorrhagic strokes. Mood disorders include but are notexcluded to conditions such as unipolar depression, bipolar depressionand anxiety. Examples of neurodegenerative diseases include amyotrophiclateral sclerosis, Parkinson's disease, acquired or symptomaticparkinsonism, Huntington's disease, and heredodegenerative disease.Examples of cognitive disorders include dementia, dementia of theAlzheimer's type, vascular dementia, multi-infarct dementia andposttraumatic dementia. Demyelinating diseases consist of diseases suchas the following: multiple sclerosis (MS), Marburg and Balo forms of MS,neuromyelitis optica, perivenous encephalitits, acute disseminatedencephalomyelitits, and acute necrotizing hemmorhhagic encephalitis.

The methods of the invention may also be applied to non-psychiatricconditions for which anti-psychotic drugs are used. Examples of theseconditions include nausea, vomiting, movement disorders associated withneurodegenerative diseases such as Huntington's disease and Tourette'ssyndrome, pruritis, and chronic hiccough. Cardiovascular disorders,cancers, neoplastic disease and other related disorders, osteoporosis,neural tube defects, neurological disorders, disorders influenced byfolic acid metabolism, metabolic or endocrine disease, inborn errors ofmetabolism, inflammation, immune disorders, psychiatric illness, andrenal disease are also relevant to these methods.

It is to be understood that the methods of the invention may be appliedto any other diseases or disorders. Examples of other diseases ordisorders may include those that are indirectly or directly affected byan increase or decrease in MTHFR activity, homocysteine levels,methionine levels, S-adenosylmethionine activity, or methylation.

The therapies relevant to the methods of the present invention includeall clinically used therapies and therapies in preclinical or clinicaldevelopment for the aforementioned diseases and disorders. Preferredtherapies include the therapies listed in databases such as Life Cycle,R&D Focus, IMS World Publications, London UK or in any other database oftherapies that are in development and/or that are FDA approved.Preferred anti-psychotic therapies include conventional neurolepticssuch as phenothiazines (e.g. chlorpromazine), thioxanthenes (e.g.thiothixene), butyrophenones (e.g. haloperidol, one of the most usefulconventional anti-psychotics), dibenzoxazepines, dibenzodiazepines,diphenylbutylpiperidines, and other heterocyclic compounds. Preferredatypical neuroleptics include compounds such as clozaril, risperidone,olanzapine, quetiapine, ziprasidone, amisulpride, sertindole, andiloperidone. Additionally, anti-psychotic therapies, such as loxapine,may have pharmacology intermediate between that of conventional andatypical neuroleptics. In preferred embodiments, the treatment orprevention of a neurological disease includes administration of one ormore therapeutically active compounds that bind to a dopamine,histamine, muscarinic cholinergic, α-adrenergic, or serotonin receptors.

Examples of other pharmaceutically active compounds which may be used asanti-psychotics include synthetic organic molecules, naturally occurringorganic molecules, nucleic acid molecules, biosynthetic proteins orpeptides, naturally occurring peptides or proteins, and modifiednaturally occurring peptides or proteins. In preferred embodiments, thepreferred therapy consists of an altered dose regime compared to otherclinically used dose regimes or to the administration of a combinationof therapeutics. It is also contemplated that the preferred therapy mayconsist of other medical treatments, such as surgical procedures,electroconvulsive therapy, or psychotherapy. Examples of psychotherapiesinclude dynamic psychotherapy, cognitive-behavioral therapy,interpersonal therapy, behavioral therapy, group psychotherapy, andfamily therapy. For the administration of a therapeutically activecompound to a subject, any mode of administration or dosing regime maybe used. It is to be understood that for any particular subject,specific dosage regimes should be adjusted over time according to theindividual need and the professional judgment of the personadministering or supervising the administration of the compositions.

Descriptions of clinically used therapies (for example, for psychosis)and guidelines for their use are readily available in common referencessuch as the Physicians Desk Reference (PDR). Additionally, therapeuticsare described and listed in medical, pharmacy, and pharmacology journalsand textbooks, such as Goodman and Gilman's The Pharamcological Basis ofTherapeutics, (Hardman and Limbird (eds.) Ninth Edition, McGraw-Hill,New York, 1996). Additional information on FDA approved therapeutics andtherapies in clinical trials for particular indications, diseases, ordisorders may be easily found in electronic databases available throughsubscription and pharmaceutical company information.

By “preferred therapy” is meant a therapy for a disease, disorder, ordysfunction that is efficacious, safe, and/or has reduced toxicitycompared to another therapy for the disease or disorder. The preferredtherapy selected based on one or more mutations in one or more MTHFRalleles is preferably effective in at least 20, 30, 50, 70, or 90% ofthe subjects having the MTHFR mutation(s). By “effective” is meantablates, reduces, or stabilizes symptoms in a subject suffering from adisease, disorder, or dysfunction prevents the onset of symptoms in asubject at risk for a disease, disorder, or dysfunction, or results in alater age-at-onset of symptoms in the subject compared to the averageage-at-onset for the corresponding untreated subjects with the sameMTHFR mutation(s). The effectiveness of the therapy for the treatment ofa psychosis may be evaluated based on the Clinical Global Impression(CGI), Diagnostic Interview for Genetic Studies (DIGS), GlobalAssessment Score (GAS), or Brief Psychiatric Rating Scale (BPRS) usingstandard procedures. Preferably, the total BPRS score is lowered by 5,more preferably 10, and most preferably 15 points. By “reduced toxicity”is meant lower level of adverse pharmacological or physiologicaleffects. Preferably, the therapy produces clinically unacceptableside-effects in 5, 10, 20, 30, or 50% fewer of the subjects having theMTHFR mutations(s) than in subjects who do not have one or ore of theMTHFR mutations.

A “polymorphism” is intended to mean a mutation or allelic variancepresent in 1% or more of alleles of the general population. Apolymorphism is disease-causing when it is present in patients with adisease but not in the general population. However, a polymorphismpresent both in patients having a disease and in the general populationis not necessarily benign. The definition of a disease-causingsubstitution, as distinct from a benign polymorphism, is based on 3factors: (1) absence of the change in at least 50 independent controlchromosomes; (2) presence of the amino acid in the bacterial enzyme,attesting to its evolutionary significance and (3) change in amino acidnot conservative. Although expression of the substitutions is requiredto formally prove that they are not benign, the criteria above allow usto postulate that the changes described in this report are likely toaffect activity.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A to 1F illustrate the first cDNA coding sequence (SEQ ID NO:1and NO:2) for methylenetetrahydrofolate reductase (MTHFR);

FIG. 2 is the alignment of amino acids for humanmethylenetetrahydrofolate reductase (MTHFR), the metF genes from E. coli(ECOMETF), and S. typhimurium (STYMETF), and an unidentified openreading frame in Saccharomyces cerevisiae that is divergentlytranscribed from an excision repair gene (ysRAD1);

FIGS. 3A and 3B illustrate the sequencing and restriction enzymeanalysis for the Arg to Ter substitution;

FIGS. 4A and 4B illustrate the sequencing and restriction enzymeanalysis for the Arg to Gln substitution;

FIGS. 5A and 5B illustrate the sequence change and restriction enzymeanalysis for the alanine to valine substitution;

FIGS. 6A to 6C illustrate the total available sequence (SEQ ID NO:3 andNO:4) of human MTHFR cDNA;

FIGS. 7A and 7B illustrate the expression analysis of MTHFR cDNA in E.coli, respectively (7A) the Western blot of bacterial extracts andtissues, and (7B) the thermolability assay of bacterial extracts;

FIGS. 8A to 8D illustrate the identification of a 5′ splice sitemutation leading to a 57-bp in-frame deletion of the cDNA;

FIGS. 9A to 9D illustrate the diagnostic restriction endonucleaseanalysis of 4 mutations;

FIGS. 10A to 10D illustrate the ASO hybridization analysis of 2mutations;

FIG. 11 illustrates the region of homology between humanmethylenetetrahydrofolate reductase (MTHFR) and human dihydrofolatereductase (DHFR);

FIGS. 12A-12B illustrate the exonic sequences of the human MTHFR genewith their flanking intronic sequences;

FIGS. 13A-13B illustrate the exonic sequences of the mouse MTHFR genewith their flanking intronic sequences;

FIG. 14 illustrates intron sizes and locations for both human and mousegenes; and

FIG. 15 illustrates the alignment of MTHFR amino acid sequences for thehuman MTHFR (hMTHFR), mouse MTHFR (MMTHFR) and the MetF gene of bacteria(bMTHFR).

DETAILED DESCRIPTION OF THE INVENTION

Sequencing of Peptides from Porcine MTHFR

Homogeneous native porcine MTHFR was digested with trypsin to generate a40 kDa N-terminal fragment and a 31 kDa C-terminal fragment; the 31 kDafragment is a proteolytic product of the 37 kDa fragment. The fragmentswere separated by SDS-PAGE, electroeluted, and the denatured fragmentswere digested with lysyl endopeptidase (LysC). The resulting peptideswere separated by reversed-phase HPLC and subjected to sequence analysisby Edman degradation (details contained in Goyette P et al., NatureGenetics, 1994, 7:195-200).

Isolation and Sequencing of cDNAs

Two degenerate oligonucleotides were synthesized based on the sequenceof a 30 amino acid porcine MTHFR peptide (first underlined peptide inFIG. 2). These were used to generate a 90 bp PCR product, encoding thepredicted peptide, from reverse transcription-PCR reactions of 500 ngpig liver polyA+ RNA. A pig-specific (non-degenerate, antisense) PCRprimer was then synthesized from this short cDNA sequence. Using thisprimer and a primer for phage arms, a human liver λgt10 cDNA library(Clontech) was screened by PCR; this technique involved the generationof phage lysate stocks (50,000 pfu) which were boiled for 5 min and thenused directly in PCR reactions with these two primers. PCR fragmentswere then sequenced directly (Cycle Sequencing™ kit, GIBCO), and apositive clone was identified by comparison of the deduced amino acidsequence to the sequence of the pig peptide (allowing for inter-speciesvariations). The positive stock was then replated at lower density andscreened with the radiolabelled positive PCR product by plaquehybridization until a well-isolated plaque was identified. Phage DNA waspurified and the insert was then subcloned into pBS+ (Bluescript) andsequenced on both strands (Cycle Sequencing™ kit, GIBCO and Sequenase™,Pharmacia). The deduced amino acid sequence of the human cDNA wasaligned to the porcine peptide sequences, the metF genes from E. coli(ecometf, accession number VO1502) and S. typhimurium (stymetF,accession number XO7689) and with a previously unidentified open readingframe in Saccharomyces cerevisiae that is divergently transcribed withrespect to the excision repair gene, ysRAD1 (accession number KO2070).The initial alignments were performed using BestFit™ in the GCG computerpackage, and these alignments were adjusted manually to maximizehomologies.

In summary, degenerate oligonucleotide primers were designed to amplifya sequence corresponding to a 30-amino acid segment of a porcine peptidefrom the N-terminal region of the enzyme (first porcine peptide in FIG.2). A 90-bp porcine cDNA fragment was obtained from reversetranscription/PCR of pig liver RNA. Sequencing of the PCR fragmentconfirmed its identity by comparison of the deduced amino acid sequenceto the porcine peptide sequence. A nondegenerate oligonucleotide primer,based on the internal sequence of the porcine cDNA, was used inconjunction with primers for the phage arms to screen a human liverλgt10 cDNA library by PCR. The insert of the positive clone was isolatedand sequenced. The sequence consisted of 1266 bp with one continuousopen reading frame.

Homology with MTHFR in Other Species

The deduced amino acid sequence of the human cDNA was aligned with themetF genes from E. coli and S. typhimurium, as well as with a previouslyunidentified ORF in Saccharomyces cerevisiae that is divergentlytranscribed with respect to the excision repair gene, ysRAD1 (FIG. 2).The sequences homologous to 5 porcine peptides are underlined in FIG. 2.Three segments (residues 61-94, 219-240, and 337-351) correspond tointernal peptide sequence from the N-terminal 40-kDa domain of theporcine liver enzyme. Residues 374-393 correspond to the upstreamportion of the LysC peptide from the C-terminal domain of the porcineliver enzyme that is labeled when the enzyme is irradiated with UV lightin the presence of (³H-methyl)AdoMet; as predicted from the AdoMetlabeling studies, this peptide lies at one end (N-terminal) of the 37kDa domain. A fifth region of homology (residues 359-372) was alsoidentified, but the localization of the porcine peptide within thenative protein had not been previously determined.

Methylenetetrahydrofolate reductase (MTHFR) is an enzyme involved inamino acid metabolism, that is critical for maintaining an adequatemethionine pool, as well as for ensuring that the homocysteineconcentration does not reach toxic levels. The high degree of sequenceconservation, from E. coli to Homo sapiens, attests to the significanceof MTHFR in these species. The enzyme in E. coil (encoded by the metFlocus) is a 33-kDa peptide that binds reduced FAD and catalyzes thereduction of methylenetetrahydrofolate to methyltetrahydrofolate. ThemetF enzyme differs from the mammalian enzyme in that NADPH or NADHcannot reduce it, and its activity is not allosterically regulated byS-adenosylmethionine. The native porcine enzyme is susceptible totryptic cleavage between the N-terminal 40 kDa domain and the C-terminal37 kDa domain, and this cleavage results in the loss of allostericregulation by adenosylmethionine, but does not result in loss ofcatalytic activity. Since the homology between the bacterial andmammalian enzymes is within the N-terminal domain, this region mustcontain the flavin binding site and residues necessary to bind thefolate substrate and catalyze its reduction. The domain structure of thehuman enzyme has not been elucidated, although the human enzyme has beenreported to have a molecular mass of 150 kDa and is likely to be ahomodimer of 77 kDa.

The predicted point of cleavage between the two domains lies betweenresidues 351 and 374 of the human sequence, based on the localization ofpeptides obtained from the isolated domains of the porcine enzyme. Thisregion, containing the highly charged sequence KRREED, is predicted tohave the highest hydrophilicity and surface probability of any region inthe deduced human sequence.

The N-terminus of the porcine protein has been sequenced, and the regionencoding this part of the protein is missing from the human cDNA. It isestimated that this cDNA is missing only a few residues at theN-terminus, since the predicted molecular mass of the deduced sequenceupstream of the putative cleavage site (KRREED) is 40 kDa, and themeasured molecular mass of the porcine N-terminal domain is also 40 kDa.When the bacterial, yeast and human sequences are aligned, the deducedhuman sequence contains an N-terminal extension of 40 amino acids; it issuspected that this extension contains determinants for NADPH binding.Many pyridine nucleotide-dependent oxidoreductases contain suchdeterminants at the N-terminus of the protein.

The C-terminus of the human sequence contains a peptide that is labeledwhen the protein is irradiated with ultraviolet light in the presence oftritiated AdoMet. The cDNA sequence reported here contains only about 7kDa of the predicted 37-kDa mass of this domain, indicating that thiscDNA is truncated at the 3′ terminus as well. A number of peptides fromthe C-terminal porcine domain have also not been detected. As might beexpected, given that the prokaryotic enzymes do not appear to beallosterically regulated by AdoMet, there are no significant homologiesbetween the C-terminal region in this cDNA and the prokaryotic metFsequences. The alignment shown in FIG. 2 shows that the homologoussequences terminate just prior to the putative cleavage site of thehuman enzyme.

Chromosomal Assignment

In situ hybridization to metaphase human chromosomes was used forlocalization of the human gene. The analysis of the distribution of 200silver grains revealed a significant clustering of grain 40 grains, inthe p36.3-36.2 region of chromosome 1 (p<0.0001), with the majority ofgrains, 25 grains, observed over 1p36.3.

The isolation of the human cDNA has allowed us to localize the gene tochromosome 1p36.3. The observation of one strong signal on thatchromosome with little background is highly suggestive of a single locuswith no pseudogenes. Southern blotting of human DNA revealed fragmentsof approximately 10 kb, predicting a gene of average size, since thiscDNA encodes approximately half of the coding sequence.

Additional cDNA Sequences and Constructs for Expression Analysis

A human colon carcinoma cDNA library (gift of Dr. Nicole Beauchemin,McGill University) was screened by plaque hybridization with theoriginal 1.3-kb cDNA to obtain additional coding sequences. A cDNA of2.2 kb was isolated, which contained 1.3 kb of overlapping sequence tothe original cDNA and 900 additional bp at the 3′ end (FIG. 6). Theamino acid sequence is identical to that of the original cDNA for theoverlapping segment (codons 1-415) except for codon 177 (ASP) which wasa GLY codon in the original cDNA. Analysis of 50 control chromosomesrevealed an ASP codon at this position. The cDNA has an open readingframe of 1980 bp, 100 bp of 3′ UTR and a poly A tail.

Sequencing was performed on both strands for the entire cDNA. Additional5′ sequences (800 bp) were obtained from a human kidney cDNA library(Clontech) but these sequences did not contain additional codingsequences and were therefore used for the PCR-based mutagenesis only (asdescribed below) and not for the expression analysis. The two cDNAs (2.2kb and 800 bp) were ligated using the EcoRI site at bp 199 and insertedinto the Bluescript™ vector (Stratagene). The 2.2 kb cDNA was subclonedinto the expression vector pTrc99A (Pharmacia) using the NcoI site at bp11 and the XbaI site in the polylinker region of both the Bluescript™and the pTrc99A vectors. Sequencing was performed across the cloningsites to verify the wild-type construct.

Utility of Invention in Identification of Mutations I. Identification ofFirst Two Mutations in Severe MTHFR Deficiency

Total RNA of skin fibroblasts from MTHFR-deficient patients wasreverse-transcribed and amplified by PCR for analysis by the singlestrand conformation polymorphism (SSCP) method (Orita, M. et al.,Genomics, 1989, 5:8874-8879). Primers were designed to generatefragments of 250-300 bp and to cover the available cDNA sequences withsmall regions of overlap for each fragment at both ends. The firstmutation identified by SSCP was a C to T substitution at bp 559 inpatient 1554; this substitution converted an arginine codon to atermination codon (FIG. 3A). Since the mutation abolished a FokI site,restriction digestion was used for confirmation of the change and forscreening additional patients for this mutation; a second patient (1627)was identified in this manner (FIG. 3B). The SSCP pattern for patient1554 and the restriction digestion pattern for both patients wasconsistent with a homozygous mutant state or with a genetic compoundconsisting of the nonsense mutation with a second mutation that did notproduce any detectable RNA (null allele). Studies in the parents arerequired for confirmation.

The second substitution (FIG. 4A) was a G to A transition at bp 482 inpatient 1834 that converted an arginine into a glutamine residue. Thesubstitution created a PstI site that was used to verify thesubstitution and to identify a second patient (1863) with this change(FIG. 4B). The SSCP analysis and the restriction digestion pattern wereconsistent with a heterozygous state for both patients. The argininecodon affected by this change is an evolutionarily conserved residue, asshown in FIG. 2. This observation, in conjunction with the fact that thecodon change is not conservative, makes a strong argument that thesubstitution is a pathologic change rather than a benign polymorphism.Furthermore, 35 controls (of similar ethnic background to that of theprobands) were tested for this substitution by Southern blotting ofPstI-digested DNA; all were negative.

The family of patient 1834 was studied. The symptomatic brother and themother of the proband were all shown to carry this substitution, whereasthe father was negative for the change (FIG. 4B). In the family of 1863,the mother of the proband was shown to be a carrier, while the fatherand an unaffected brother were negative.

Cell Lines

Cell line 1554 is from a Hopi male who was admitted at age three monthswith homocystinuria, seizures, dehydration, corneal clouding, hypotoniaand Candida sepsis. Folate distribution in cultured fibroblasts showed aPediococcus cerivisiae/Lactobacillus casei (PC/LC) ratio of 0.52(Control 0.14). There was no measurable methylenetetrahydrofolatereductase (MTHFR) activity (Control values=9.7 and 15.1 nmoles/h/mgprotein; residual activity after treatment of control extracts at 55° C.for 20 min.=28% and 31%).

Cell line 1627 is from a Choctaw male who presented with poor feeding,apnea, failure to thrive, dehydration and homocystinuria at five weeksof age. He was subsequently found to have superior sagittal sinusthrombosis and hydrocephalus. The PC/LC ratio was 0.61 and the specificactivity of MTHFR was 0.1 nmoles/h/mg protein. There is consanguinity inthat the maternal and paternal grandmothers are thought to be “distantlyrelated”.

Cell line 1779 is from a French Canadian male with homocystinuria whofirst had limb weakness, uncoordination, paresthesiae, and memory lapsesat age 15 years, and was wheelchair-bound in his early twenties. Hisbrother (cell line 1834) also has homocystinuria, but is 37 years oldand asymptomatic. Specific activity of MTHFR was 0.7 and 0.9 nmole/h/mgprotein for 1779 and 1834, respectively; the residual activity afterheat treatment at 55° C. was 0.9% and 0% for 1779 and 1834,respectively.

Cell line 1863 is from a white male who was diagnosed at age 21 yearsbecause of a progressive gait disturbance, spasticity, cerebral whitematter degeneration, and homocystinuria. He had a brother who died atage 21 years of neurodegenerative disease. Specific activity of MTHFR infibroblast extracts was 1.76 nmoles/h/mg protein and the residual enzymeactivity after treatment at 55° C. was 3.6%.

Mutation Analysis

Primers were designed from the cDNA sequence to generate 250-300 bpfragments that overlapped 50-75 bp at each end. The primer pairs wereused in reverse transcription-PCR of 5 μg patient total fibroblast RNA.The PCR products were analyzed by a non-isotopic rapid SSCP protocol(PhastSystem™, Pharmacia), which uses direct silver staining fordetection of single strands. Any PCR products from patients showing ashift on SSCP gels were purified by NuSieve (FMC Bioproducts) andsequenced directly (Cycle Sequencing™ kit, GIBCO) to identify thechange. If the change affected a restriction site, then a PCR productwas digested with the appropriate restriction endonuclease and analyzedon polyacrylamide gels. To screen for the Arg to Gln mutation incontrols, 5 μg of PstI-digested DNA was run on 0.8% agarose gels andanalyzed by Southern blotting using the radiolabelled cDNA by standardtechniques.

II. Seven Additional Mutations at the MethylenetetrahydrofolateReductase (MTHFR) Locus with Genotype: Phenotype Correlation in SevereMTHFR Deficiency It is reported hereinbelow the characterization of 7additional mutations at this locus: 6 missense mutations and a 5′ splicesite defect which activates a cryptic splice site in the codingsequence. A preliminary analysis of the relationship between genotypeand phenotype for all 9 mutations identified thus far at this locus isalso reported. A nonsense mutation and 2 missense mutations (proline toleucine and threonine to methionine) in the homozygous state areassociated with extremely low activity (0-3%) and onset of symptomswithin the first year. Other missense mutations (arginine to cysteineand arginine to glutamine) are associated with higher enzyme activityand later onset of symptoms.

7 additional mutations at the MTHFR locus are described and theassociation between genotype, enzyme activity, and clinical phenotype insevere MTHFR deficiency is examined.

Patient Description

The clinical and laboratory findings of the patients have been reportedin the published literature. Residual MTHFR activity was previouslymeasured in cultured fibroblasts at confluence.

Patient 354, an African-American girl, was diagnosed at age 13 yearswith mild mental retardation. Her sister, patient 355 was diagnosed atage 15 years with anorexia, tremor, hallucinations and progressivewithdrawal. In patient 354, residual MTHFR activity was 19% and in hersister, 355, it was 14% of control values. The residual activity afterheating had equivalent thermal stability to control enzyme.

Patient 1807, a Japanese girl whose parents are first cousins, haddelayed walking and speech until age 2 years, seizures at age 6 yearsand a gait disturbance with peripheral neuropathy at age 16 years.Residual activity of MTHFR was 3% and the enzyme was thermolabile.

Patient 735, an African-Indian girl, was diagnosed at age 7 months withmicrocephaly, progressive deterioration of mental development, apnea andcoma. Residual activity of MTHFR was 2% of control levels. Thermalproperties were not determined.

Patient 1084, a Caucasian male, was diagnosed at age 3 months with aninfantile fibrosarcoma. He was found to be hypotonic and became apneic.He died at the age of 4 months. Residual activity of MTHFR was notdetectable. Thermal properties were not determined.

Patient 356, the first patient reported with MTHFR deficiency, is anItalian-American male who presented at age 16 years with muscleweakness, abnormal gait and flinging movements of the upper extremities.MTHFR residual activity was 20% of control values; activity was rapidlyand exponentially inactivated at 55°.

Patient 458, a Caucasian male, was diagnosed at age 12 years with ataxiaand marginal school performance. Residual MTHFR activity wasapproximately 10%, and the activity was thermolabile.

Patient 1396, a Caucasian female, was described as clumsy and as havinga global learning disorder in childhood. At age 14 years, she developedataxia, foot drop, and inability to walk. She developed deep veinthrombosis and bilateral pulmonary emboli. Residual activity of MTHFRwas 14% and the enzyme was thermolabile.

Genomic Structure and Intronic Primers

Exon nomenclature is based on available cDNA sequence in Goyette et al.(Nature Genetics, 1994, 7:195-200). Exon 1 has been arbitrarilydesignated as the region of cDNA from bp 1 to the first intron.Identification of introns was performed by amplification of genomic DNAusing cDNA primer sequences. PCR products that were greater in size thanexpected cDNA sizes were sequenced directly.

Mutation Detection

Specific exons (see Table 1 for primer sequences) were amplified by PCRfrom genomic DNA and analyzed by the SSCP protocol. SSCP was performedwith the Phastgel™ system (Pharmacia), a non-isotopic rapid SSCPprotocol, as previously described (Goyette P et al., Nature Genetics,1994, 7:195-200), or with ³⁵S-labeled PCR products run on 6% acrylamide:10% glycerol gels at room temperature (6 watts, overnight). In somecases, the use of restriction endonucleases, to cleave the PCR productbefore SSCP analysis, enhanced the detection of band shifts. PCRfragments with altered mobility were sequenced directly (GIBCO, CycleSequencing™ kit). If the sequence change affected a restrictionendonuclease site, then the PCR product was digested with theappropriate enzyme and analyzed by PAGE. Otherwise, allele-specificoligonucleotide (ASO) hybridization was performed on a dot blot of thePCR-amplified exon.

TABLE 1 PCR Primers for DNA amplification and mutation analysis of MTHFRFrag- ment Primer Size Exon Type Primer Sequence (5′→3′) Location (bp) 1Sense AGCCTCAACCCCTGCTTGGAGG C 271 (SEQ ID NO: 5) Anti-TGACAGTTTGCTCCCCAGGCAC I sense (SEQ ID NO: 6) 4 SenseTGAAGGAGAAGGTGTCTGCGGGA C 198 (SEQ ID NO: 7) Anti- AGGACGGTGCGGTGAGAGTGGI sense (SEQ ID NO: 8) 5 Sense CACTGTGGTTGGCATGGATGATG I 392 (SEQ ID NO:9) Anti- GGCTGCTCTTGGACCCTCCTC I sense (SEQ ID NO: 10) 6 SenseTGCTTCCGGCTCCCTCTAGCC I 251 (SEQ ID NO: 11) Anti- CCTCCCGCTCCCAAGAACAAAGI sense (SEQ ID NO: 12)

TABLE 2 Summary of genotypes, enzyme activity, age at onset, andbackground of patients with MTHFR deficiency Amino acid % Patient^(a)BPChanges^(b) changes Activity Age at Onset Background 1807 C764T/C764TPro→Leu/Pro→Leu 3 within 1st year Japanese  735 C692T/C692TThr→Met/Thr→Met 2  7 months African Indian 1084 C692T/C692TThr→Met/Thr→Met 0  3 months Caucasian 1554 C559T/C559T Arg→Ter/Arg→Ter 0 1 month Native American (Hopi) 1627 C559T/C559T Arg→Ter/Arg→Ter 1  1month Native American (Choctaw)  356 C985T/C985T Arg→Cys/Arg→Cys 20 16yrs Italian American  458 C1015T/G167A Arg→Cys/Arg→Gln 10 11 yrsCaucasian 1396 C1081T/G167A Arg→Cys/Arg→Gln 14 14 yrs Caucasian 1779^(c)G482A/? Arg→Gln/? 6 15 yrs French Canadian 1834^(c) G482A/? Arg→Gln/? 7Asymptomatic French Canadian at 37 yrs 1863 G482A/? Arg→Gln/? 14 21 yrsCaucasian  354^(d) 792 + 1G→A/? 5′ splice site/? 19 13 yrs AfricanAmerican  355^(d) 792 + 1G→A/? 5′ splice site/? 14 11 yrs AfricanAmerican ^(a)Patients 1554, 1627, 1779, 1834 and 1863 were previouslyreported by Goyette et al. (1994) ^(b)→ = unidentified mutation.^(c)Patients 1779 and 1834 are sibs. ^(d)Patients 354 and 355 are sibs.

(1) 5′ Splice Site Mutation

Amplification of cDNA, bp 653-939, from reverse-transcribed totalfibroblast RNA revealed 2 bands in sisters 354 and 355: a smaller PCRfragment (230 bp) in addition to the normal 287 bp allele (FIG. 8A).FIG. 8A is the PAGE analysis of amplification products of cDNA bp653-939, from reverse transcribed RNA. Controls have the expected 287-bpfragment while patients 354 and 355 have an additional 230-bp fragment.Sequencing of the smaller fragment identified a 57-bp in-frame deletionwhich would remove 19 amino acids (FIG. 8B). FIG. 8B is the directsequencing of the PCR products from patient 354. The 57-bp deletionspans bp 736-792 of the cDNA. An almost perfect 5′ splice site (boxed)is seen at the 5′ deletion breakpoint. Analysis of the sequence at the5′ deletion breakpoint in the undeleted fragment revealed an almostperfect 5′ splice site consensus sequence (AG/gcatgc). This observationsuggested the presence of a splicing mutation in the natural 5′ splicesite that might activate this cryptic site, to generate the deletedallele. The sequence following the deletion breakpoint, in the mutantallele, corresponded exactly to the sequence of the next exon.Amplification of genomic DNA, using the same amplification primers asthose used for reverse-transcribed RNA, generated a 1.2-kb PCR productindicating the presence of an intron. Direct sequencing of this PCRfragment in patient 354 identified a heterozygous G→A substitution inthe conserved GT dinucleotide of the intron at the 5′ splice site (FIG.8C). FIG. 8C is the sequencing of the 5′ splice site in control andpatient 354. The patient carries a heterozygous G→A substitution in the5′ splice site (boxed). Intronic sequences are in lower case. Thissubstitution abolished a HphI restriction endonuclease site which wasused to confirm the mutation in the 2 sisters (FIG. 8D). FIG. 8D is theHphI restriction endonuclease analysis on PCR products of DNA for exon 4of patients 354 and 355, and of 3 controls (C). The 198-bp PCR producthas 2 HphI sites. The products of digestion for the control allele are151, 24 and 23 bp. The products of digestion for the mutant allele are175 and 23 bp due to the loss of a HphI site. The fragments of 24 and 23bp have been run off the gel.

(2) Patients with Homozygous Coding Substitutions

SSCP analysis of exon 4 for patient 1807 revealed anabnormally-migrating fragment, which was directly sequenced to reveal ahomozygous C→T substitution (bp 764) converting a proline to a leucineresidue. This change creates a MnlI restriction endonuclease site, whichwas used to confirm the homozygous state of the mutation (FIG. 9A). FIG.9A is the MnlI restriction analysis of exon 4 PCR products for patient1807 and 3 controls (C). Expected fragments: control allele, 90, 46, 44,18 bp; mutant allele, 73, 46, 44, 18, 17 bp. An additional band at thebottom of the gel is the primer. Fifty independent control Caucasianchromosomes and 12 control Japanese chromosomes were tested byrestriction analysis; all were negative for this mutation. Homozygosityin this patient is probably due to the consanguinity of the parents.

Patients 735 and 1084 had the same mutation in exon 4, in a homozygousstate: a C→T substitution (bp 692) which converted an evolutionarilyconserved threonine residue to a methionine residue, and abolished aNlaIII restriction endonuclease site. Allele-specific oligonucleotidehybridization to amplified exon 4 (FIGS. 10A and 10B) was used toconfirm the mutation in these 2 patients and to screen 60 independentchromosomes, all of which turned out to be negative. FIG. 10A is thehybridization of mutant oligonucleotide (692T) to exon 4 PCR productsfrom patients 735, 1084 and 30 controls. Only DNA from patients 735 and1084 hybridized to this probe. FIG. 10B is the hybridization of normaloligonucleotide (692C) to stripped dot blot from A. All control DNAshybridized to this probe.

Patient 356 showed a shift on SSCP analysis of exon 5. Direct sequencingrevealed a homozygous C→T substitution (bp 985) which converted anevolutionarily conserved arginine residue to cysteine; the substitutionabolished an AciI restriction endonuclease site. This was used toconfirm the homozygous state of the mutation in patient 356 (FIG. 9B)and its presence in the heterozygous state in both parents. Fiftyindependent control chromosomes, tested in the same manner, werenegative for this mutation. FIG. 9B is the AciI restriction analysis ofexon 5 PCR products for patient 356, his father (F), his mother (M), and3 controls (C). Expected fragments: control allele, 129, 105, 90, 68 bp;mutant allele, 195, 129, 68 bp.

(3) Patients who are Genetic Compounds

Patient 458 is a compound heterozygote of a mutation in exon 5 and amutation in exon 1. The exon 5 substitution (C→T at bp 1015) resulted inthe substitution of a cysteine residue for an arginine residue; thisabolished a HhaI restriction endonuclease site, which was used toconfirm the mutation in patient 458 (FIG. 9C) and to show that 50control chromosomes were negative. FIG. 9C is the HhaI restrictionanalysis of exon 5 PCR products for patient 458 and 4 controls (C).Expected fragments: control allele, 317 and 75 bp; mutant allele 392 bp.The 75-bp fragment is not shown in FIG. 9C. The second mutation was aheterozygous G→A substitution (bp 167) converting an arginine to aglutamine residue. Allele-specific oligonucleotide hybridization toamplified exon 1 confirmed the heterozygous state of this mutation inpatient 458 and identified a second patient (1396) carrying thismutation also in the heterozygous state (FIGS. 10C and 10D). FIG. 10C isthe hybridization of mutant oligonucleotide (167A) to exon 1 PCRproducts from patients 458, 1396 and 31 controls. FIG. 10D is thehybridization of normal oligonucleotide (167G) to stripped dot blot fromC. None of the 62 control chromosomes carried this mutation.

The second mutation in patient 1396 was identified in exon 6: aheterozygous C→T substitution (bp 1081) that converted an arginineresidue to a cysteine residue, and abolished a HhaI restrictionendonuclease site. Restriction analysis confirmed the heterozygoussubstitution in 1396 (FIG. 9D) and showed that 50 control chromosomeswere negative. Tooth decay does not occur in patients having a salivaabove pH 5.0. FIG. 9D is the HhaI restriction analysis of exon 6 PCRproducts for patient 1396 and 2 controls (C). Expected fragments:control allele, 152, 86, 13 bp; mutant allele 165, 86 bp. The 13-bpfragment has been run off the gel.

(4) Additional Sequence Changes

HhaI analysis of exon 6, mentioned above, revealed a DNA polymorphism.This change is a T→C substitution at bp 1068 which does not alter theamino acid (serine), but creates a HhaI recognition site. T at bp 1068was found in 9% of tested chromosomes. Sequence analysis identified 2discrepancies with the published cDNA sequence: a G→A substitution at bp542 which converts the glycine to an aspartate codon, and a C→T changeat bp 1032 which does not alter the amino acid (threonine). Since allDNAs tested (>50 chromosomes) carried the A at bp 542 and the T at bp1032, it is likely that the sequence of the original cDNA contained somecloning artifacts.

Genotype:Phenotype Correlation

Table 2 summarizes the current status of mutations in severe MTHFRdeficiency. In 8 patients, both mutations have been identified; in 5patients (3 families), only 1 mutation has been identified. Overall thecorrelation between the genotype, enzyme activity, and phenotype isquite consistent. Five patients, with onset of symptoms within the firstyear of life, had ≦3% of control activity. Three of these patients hadmissense mutations in the homozygous state: two patients with thethreonine to methionine substitution (C692T) and one patient with theproline to leucine substitution (C764T). The nonsense mutation (C559T)in the homozygous state in patients 1554 and 1627 (previously reportedin Goyette P et al., Nature Genetics, 1994, 7:195-200) is alsoassociated with a neonatal severe form, as expected.

The other patients in Table 2 had ≧6% of control activity and onset ofsymptoms within or after the 2nd decade of life; the only exception ispatient 1834, as previously reported (Goyette P et al., Nature Genetics,1994, 7:195-200). The three patients (356, 458 and 1396) with missensemutations (G167A, C985T, C1015T and C1081T) are similar to thosepreviously reported (patients 1779, 1834 and 1863) who had an arginineto glutamine substitution and a second unidentified mutation (Goyette Pet al., Nature Genetics, 1994, 7:195-200). The sisters with the 5′splice mutation and an unidentified second mutation also had levels ofactivity in the same range and onset of symptoms in the second decade,but the activity is likely due to the second unidentified allele.

Discussion

The patients come from diverse ethnic backgrounds. Although patients1554 and 1627 are both Native Americans, the mutations occur ondifferent haplotypes, suggesting recurrent mutation rather than identityby descent. Since the substitution occurs in a CpG dinucleotide, a “hotspot” for mutation, recurrent mutation is a reasonable hypothesis. It isdifficult to assess whether some mutations are population-specific sincethe numbers are too small at the present time.

MTHFR deficiency is the most common inborn error of folate metabolism,and a major cause of hereditary homocysteinemia. The recent isolation ofa cDNA for MTHFR has permitted mutational analysis at this locus, withthe aims of defining important domains for the enzyme and of correlatinggenotype with phenotype in MTHFR-deficient patients.

The 7 mutations described here (6 single amino acid substitutions and a5′ splice site mutation) bring the total to 9 mutations identified thusfar in severe MTHFR deficiency and complete the mutation analysis for 8patients. The identification of each mutation in only one or twofamilies points to the striking degree of genetic heterogeneity at thislocus. Seven of the 9 mutations are located in CpG dinucleotides, whichare prone to mutational events.

5′ Splice Site Mutation

The G→A substitution at the GT dinucleotide of the 5′ splice site inpatients 354 and 355 results in a 57bp in-frame deletion of the codingsequence, which should delete 19 amino acids of the protein. Thisdeletion occurs as a result of the activation of a cryptic 5′ splicesite (AG/gc) even though this cryptic site does not have a perfect 5′splice site consensus sequence (AG/gt). However, GC (instead of GT) asthe first 2 nucleotides of an intron has been reported in severalnaturally-occurring splice sites, such as in the genes for humanprothrombin and human adenine phosphoribosyltransferase and twice withinthe gene for the largest subunit of mouse RNA polymerase II. Theremaining nucleotides of the cryptic site conform to a normal splicesite consensus sequence with its expected variations (A₆₀G₇₉/g₁₀₀t₁₀₀a₅₉a₇₁g₈₂t₅₀). It is unlikely that the deleted enzymeresulting from this aberrantly-spliced mRNA would have any activity; 8of the 19 deleted amino acids are conserved in the E. coli enzyme.Although the 2 patients show residual enzyme activity in the range of20% of controls, the activity is probably due to the unidentified secondallele in these patients.

6 Missense Mutations

The Arg→Cys substitution (C1081T) in patient 1396 is within ahydrophilic sequence previously postulated to be the linker regionbetween the catalytic and regulatory domains of MTHFR (Goyette P et al.,Nature Genetics, 1994, 7:195-200). These 2 domains are readily separableby mild trypsinization of the porcine enzyme. The linker domain, ahighly-charged region, is likely to be located on the outside surface ofthe protein and therefore more accessible to proteolysis. Because theArg→Cys substitution converts a charged hydrophilic residue to anuncharged polar residue, it cannot be considered a conservative change,and could affect the stability of the enzyme.

The 2 Arg→Cys substitutions identified in patients 356 and 458 (C985Tand C1015T, respectively) may be involved in binding the FAD cofactor.Previous work in the literature showed that heating fibroblast extractsat 55°, in the absence of the FAD cofactor, inactivated MTHFRcompletely. The addition of FAD to the reaction mixture before heatinactivation restored some enzyme activity to control extracts and toextracts from some patients, while the extracts of patients 356 and 458were unaffected. Based on these observations, it was suggested thatthese 2 patients had mutations affecting a region of the proteininvolved in binding FAD. The 2 mutations are found in close proximity toeach other, within 11 amino acids. In patient 356, the Arg residue isevolutionarily-conserved in E. coli and is found in a stretch of 9conserved amino acids, suggesting a critical role for this residue; thealtered Arg residue in patient 458 is not evolutionarily-conserved.Crystal structure analysis of medium chain acyl-CoA dehydrogenase(MCAD), a flavoprotein, has defined critical residues involved in thebinding of FAD. Two consecutive residues of the MCAD protein, Met165 andTrp166, involved in interactions with FAD, can also be identified inMTHFR, 3 and 4 amino acids downstream, respectively, from the Argresidue altered in patient 458.

The Thr→Met substitution (C692T) is found in a region of highconservation with the E. coli enzyme and in a region of good homologywith human dihydrofolate reductase (DHFR) (FIG. 11). In FIG. 11, =isidentity;  is homology; and $ is identity to bovine DHFR enzyme. Anasterisk (*) indicates location of Thr→Met substitution. Considering theearly-onset phenotype of the patients, one can assume that the threonineresidue is critical for activity or that it contributes to an importantdomain of the protein. This region of homology in DHFR contains aresidue, Thr136, which has been reported to be involved in folatebinding. This Thr residue in DHFR aligns with a Ser residue in MTHFR, anamino acid with similar biochemical properties. The Thr→Met substitutionis located 8 amino acids downstream from this Ser codon, in the centerof the region of homology between the 2 enzymes. It is thereforehypothesized that the Thr→Met substitution may alter the binding of thefolate substrate.

The G167A (Arg→Gln) and C764T (Pro→Leu) substitutions both affectnon-conserved amino acids. Their importance in the development of MTHFRdeficiency cannot be determined at the present time. All the mutationsidentified thus far are located in the 5′ end of the coding sequence,the region thought to encode the catalytic domain of MTHFR. Mutationanalysis has been useful in beginning to address the structure: functionproperties of the enzyme as well as to understand the diverse phenotypesin severe MTHFR deficiency.

III. Identification of A→V Mutation

SSCP analysis and direct sequencing of PCR fragments were used toidentify a C to T substitution at bp 677, which converts an alanineresidue to a valine residue (FIG. 5A). The primers for analysis of theA→V change are: 5′-TGAAGGAGAA GGTGTCTGCG GGA-3′ (SEQ ID NO:13) (exonic)and 5′-AGGACGGTGC GGTGAGAGTG-3′ (SEQ ID NO:14) (intronic); these primersgenerate a fragment of 198 bp. FIG. 5A depicts the sequence of twoindividuals, a homozygote for the alanine residue and a homozygote forthe valine residue. The antisense strands are depicted. This alterationcreates a HinfI site (FIG. 5B), which was used to screen 114 unselectedFrench Canadian chromosomes; the allele frequency of the substitutionwas 0.38. The substitution creates a HinfI recognition sequence whichdigests the 198 bp fragment into a 175 bp and a 23 bp fragment; thelatter fragment has been run off the gel. FIG. 5B depicts the threepossible genotypes. The frequency of the 3 genotypes were as follows:−/−, 37%; ±, 51%; and +/+, 12% (the (+) indicates the presence of theHinfI restriction site and a valine residue).

The alanine residue is conserved in porcine MTHFR, as well as in thecorresponding metF and stymetF genes of E. coli and S. typhimurium,respectively. The strong degree of conservation of this residue, and itslocation in a region of high homology with the bacterial enzymes,alluded to its importance in enzyme structure or function. Furthermore,the frequency of the (+/+) genotype was consistent with the frequency ofthe thermolabile MTHFR variant implicated in vascular disease.

Clinical Material

To determine the frequency of the A→V mutation, DNA from 57 individualsfrom Quebec was analyzed by PCR and restriction digestion. Theindividuals, who were all French Canadian, were not examined clinicallyor biochemically.

The 40 individuals analyzed in Table 3 had been previously described inEngbersen et al. (Am. J. Hum. Genet., 1995, 56:142-150). Of the 13cardiovascular patients, 8 had cerebrovascular arteriosclerosis and 5had peripheral arteriosclerosis. Five had thermolabile MTHFR while 8 hadthermostable MTHFR (greater than 33% residual activity after heating).Controls and patients were all Dutch-Caucasian, between 20-60 years ofage. None of these individuals used vitamins which could alterhomocysteine levels. Enzyme assays and homocysteine determinations werealso reported by Engbersen et al. (Am. J. Hum. Genet., 1995,56:142-150).

TABLE 3 Correlation between MTHFR genotype and enzyme activity,thermolability and plasma homocysteine level −/− +/− +/+ n = 19 n = 9 n= 12 specific activity^(a,b) 22.9 ± 1.7 15.0 ± 0.8 6.9 ± 0.6 (nmol CH₂0/(11.8-33.8) (10.2-18.8)  (2.6-10.2) mg · protein/hr) residual activityafter 66.8 ± 1.5 56.2 ± 2.8 21.8 ± 2.8  heating^(a,b) (%) (55-76)(41-67) (10-35) plasma homocysteine^(a,c) 12.6 ± 1.1 13.8 ± 1.0 22.4 ±2.9  (μM)(after fasting)  (7-21) (9.6-20)  (9.6-42)  plasmahomocysteine^(a,c)  41.3 ± 5.0^(d)   41 ± 2.8  72.6 ± 11.7^(e)(μM)(post-methionine load) (20.9-110)  (29.1-54)   (24.4-159) ^(a)one-way anova p < .01 ^(b)paired t test for all combinations p < .01^(c)paired t test p < .05 for +/+ group versus +/− group or −/− group;p > .05 for +/− versus −/− group. ^(d)n = 18 for this parameter ^(e)n =11 for this parameter

Enzyme activity and plasma homocysteine were determined as previouslyreported. Each value represents mean±standard error. The range is givenin parentheses below the mean.

Correlation of A→V Mutation with Altered MTHFR Function

A genotypic analysis was performed and enzyme activity andthermolability were measured in a total of 40 lymphocyte pellets frompatients with premature vascular disease and controls. 13 vascularpatients were selected from a previous study (Engbersen et al., Am. J.Hum. Genet., 1995, 56:142-150), among which 5 were considered to havethermolabile MTHFR. From a large reference group of 89 controls, all 7individuals who had thermolabile MTHFR were studied, and an additional20 controls with normal MTHFR were selected from the same referencegroup. Table 3 documents the relationship between genotypes and specificenzyme activity, thermolability and plasma homocysteine level. The meanMTHFR activity for individuals homozygous for the substitution (+/+) wasapproximately 30% of the mean activity for (−/−) individuals, homozygousfor the alanine residue. Heterozygotes had a mean MTHFR activity thatwas 65% of the activity of (−/−) individuals; this value is intermediatebetween the values for (−/−) and (+/+) individuals. The ranges ofactivities showed some overlap for the heterozygous and (−/−) genotypes,but homozygous (+/+) individuals showed virtually no overlap with theformer groups. A one-way analysis of variance yielded a p value<0.0001;a pairwise Bonferroni t test showed that all three genotypes weresignificantly different with p<0.01 for the three possible combinations.

The three genotypes were all significantly different (p<0.01) withrespect to enzyme thermolability. The mean residual activity after heatinactivation for 5 minutes at 46° was 67%, 56% and 22% for the (−/−),(±) and (+/+) genotypes, respectively. While the degree ofthermolability overlaps somewhat for (−/−) individuals andheterozygotes, individuals with two mutant alleles had a distinctlylower range. Every individual with the (+/+) genotype had residualactivity<35% after heating, and specific activity<50% of that of the(−/−) genotype.

Total homocysteine concentrations, after fasting and 6 hours aftermethionine loading, were measured in plasma by high performance liquidchromatography using fluorescence detection. Fasting homocysteine levelsin (+/+) individuals were almost twice the value for (±) and (−/−)individuals. The differences among genotypes for plasma homocysteinewere maintained when homocysteine was measured following 6 hours ofmethionine loading. A one-way anova yielded a p<0.01 for the fasting andpost-methionine homocysteine levels. A pairwise Bonferroni t test showedthat only homozygous mutant individuals had significantly elevatedhomocysteine levels (p<0.05).

PCR-Based Mutagenesis for Expression of A→V Mutation in Vitro

PCR-based mutagenesis, using the cDNA-containing Bluescript™ vector astemplate, was used to create the A to V mutation. Vent™ polymerase (NEB)was used to reduce PCR errors. The following primers were used: primer1, bp −200 to −178, sense; primer 2, bp 667 to 687, antisense,containing a mismatch, A, at bp 677; primer 3, 667 to 687, sense,containing a mismatch, T, at bp 677; primer 4, bp 1092 to 1114,antisense. PCR was performed using primers 1 and 2 to generate a productof 887 bp, and using primers 3 and 4 to generate a product of 447 bp.The two PCR fragments were isolated from a 1.2% agarose gel byGeneclean™ (BIO 101). A final PCR reaction, using primers 1 and 4 andthe first 2 PCR fragments as template, was performed to generate a 1.3kb band containing the mutation. The 1.3 kb fragment was digested withNcoI and MscI, and inserted into the wild-type cDNA-containingexpression vector by replacing the sequences between the NcoI site at bp11 and the MscI site at bp 943. The entire replacement fragment and thecloning sites were sequenced to verify that no additional changes wereintroduced by PCR.

Expression Analysis of Wild-Type and Mutagenized cDNA

Overnight cultures of JM105™ containing vector alone, vector+wild-typeMTHFR cDNA, or vector+mutagenized cDNA were grown at 37° C. in 2×YTmedia with 0.05 mg/ml ampicillin. Fresh 10 ml. cultures of each wereinoculated with approximately 50 μL of overnight cultures for a startingO.D. of 0.05, and were grown at 37° C. to an O.D. of 1 at 420 nM.Cultures were then induced for 2 hrs. with 1 mM IPTG and pelleted. Thecells were resuspended in TE buffer with 2 μg/ml aprotinin and 2 μg/mlleupeptin (3.5×wet weight of cells). Cell suspensions were sonicated onice for 3×15 sec. to break open cell membranes and then centrifuged for30 min. at 4° C. to pellet cell debris and unlysed cells. Thesupernatant was removed and assayed for protein concentration with theBio-Rad™ protein assay. Western analysis was performed using theAmersham ECL™ kit according to the instructions of the supplier, usingantiserum generated against purified porcine liver MTHFR. Enzymaticassays were performed by established procedures; pre-treating theextracts at 46° C. for 5 min. before determining activity assessedthermolability. Specific activities (nmol formaldehyde/hr./mg. protein)were calculated for the 2 cDNA-containing constructs after subtractionof the values obtained with vector alone (to subtract background E. coliMTHFR activity).

The MTHFR cDNA (2.2 kb) (FIG. 6) has an open reading frame of 1980 bp,predicting a protein of 74.6 kDa. The purified porcine liver enzyme hasbeen shown to have subunits of 77 kDa. Western analysis (FIG. 7A) ofseveral human tissues and of porcine liver has revealed a polypeptide of77 kDa in all the studied tissues, as well as an additional polypeptideof approximately 70 kDa in human fetal liver and in porcine liver,suggesting the presence of isozymes. Two μg of bacterial extract proteinwas used for lanes 1-3. The tissues (lanes 4-8) were prepared byhomogenization in 0.25M sucrose with protease inhibitors (2 μg/ml eachof aprotinin and leupeptin), followed by sonication (3×15 sec.) on ice.The extracts were spun for 15 min. in a microcentrifuge at 14,000 g and100 μg of supernatant protein was used for Western analysis. h=human;p=porcine.

The wild-type cDNA and a mutagenized cDNA, containing the A→Vsubstitution, were expressed in E. coli to yield a protein ofapproximately 70 kDa (FIG. 7A), which co-migrates with the smallerpolypeptide mentioned above. Treatment of extracts at 46° C. for 5minutes revealed that the enzyme containing the substitution wassignificantly more thermolabile than the wild-type enzyme (p<0.001; FIG.7B). Two separate experiments (with 3-4 replicates for each constructfor each experiment) were performed to measure thermostable activity ofthe wild-type MTHFR and mutagenized MTHFR A→V cDNAs. The values shownrepresent mean±standard error for each experiment, as % of residualactivity after heating. The means of the specific activities beforeheating (expressed as nmol formaldehyde/hr./mg. protein) were asfollows: Exp. 1, 3.8 and 5.3 for MTHFR and MTHFR A→V, respectively; Exp.2, 6.2 and 7.5 for MTHFR and MTHFR A→V, respectively. The expressionexperiments were not designed to measure differences in specificactivity before heating, since variation in efficiencies of expressioncould contribute to difficulties in interpretation. Curiously though,the specific activity for the mutant construct was higher in bothexperiments. It is possible that the mutant protein has increasedstability in E. coli, or that inclusion bodies in the extractscontributed to differences in recovery of properly-assembled enzyme.

These studies have identified a common substitution in the MTHFR genewhich results in thermolability in vitro and in vivo. The mutation, inthe heterozygous or homozygous state, correlates with reduced enzymeactivity and increased thermolability of MTHFR in lymphocyte extracts. Asignificant elevation in plasma homocysteine was observed in individualswho were homozygous for the mutation. Statistically-significantdifferences for homocysteine levels were not observed betweenheterozygotes and (−/−) individuals; this observation is not surprising,since plasma homocysteine can be influenced by several environmentalfactors, including intake of folate, vitamin B₁₂, vitamin B₆, andmethionine, as well as by genetic variation at other loci, such as thecystathionine-β-synthase gene.

The alanine to valine substitution conserves the hydrophobicity of theresidue and is associated with small changes in activity, in contrast tonon-conservative changes, such as the previously-reported arginine toglutamine change in MTHFR, which is associated with a greater decreasein enzyme activity and severe hyperhomocysteinemia. The alanine residueis situated in a region of homology with the bacterial metF genes. Thesame region of homology was also observed in the human dihydrofolatereductase (DHFR) gene (FIG. 11), although the alanine residue itself isnot conserved; this region of amino acids 130-149 of DHFR contains T136which has been implicated in folate binding in an analysis of thecrystal structure of recombinant human DHFR. It is tempting to speculatethat this region in MTHFR is also involved in folate binding and thatthe enzyme may be stabilized in the presence of folate. This hypothesisis compatible with the well-documented influence of folate onhomocysteine levels and with the reported correction of mildhyperhomocysteinemia by folic acid in individuals with prematurevascular disease, and in individuals with thermolabile MTHFR.

Although the cDNA is not long enough to encode the larger MTHFRpolypeptide, it is capable of directing synthesis of the smallerisozyme. The ATG start codon for this polypeptide is within a goodconsensus sequence for translation initiation. Whether the isozyme isrestricted to liver and what its role is in this tissue remain to bedetermined.

These data have identified a common genetic change in MTHFR whichresults in thermolability; these experiments do not directly address therelationship between this change and vascular disease. Nonetheless, thispolymorphism represents a diagnostic test for evaluation of MTHFRthermolability in hyperhomocysteinemia. Large case-control studies arerequired to evaluate the frequency of this genetic change in variousforms of occlusive arterial disease and to examine the interactionbetween this genetic marker and dietary factors. Well-definedpopulations need to be examined, since the limited data set thus farsuggests that population-specific allele frequencies may exist. Moreimportantly, however, the identification of a candidate genetic riskfactor for vascular disease, which may be influenced by nutrient intake,represents a critical step in the design of appropriate therapies forthe homocysteinemic form of arteriosclerosis.

cDNA for MTHFR and its Potential Utility

The cDNA sequence is a necessary starting point for the detection ofMTHFR sequence abnormalities that would identify individuals at risk forcardiovascular and neurological diseases, as well as other disordersaffected by folic acid metabolism. Diagnostic tests by DNA analysis aremore efficient and accurate than testing by enzymatic/biochemicalassays. Less blood is required and results are available in a shorterperiod of time. The tests could be performed as a routine operation inany laboratory that performs molecular genetic diagnosis, without thespecialized reagents/expertise that is required for an enzyme-basedtest.

The second major utility of the cDNA would be in the design oftherapeutic protocols, for correction of MTHFR deficiency. Theseprotocols could directly involve the gene, as in gene therapy trials orin the use of reagents that could modify gene expression. Alternatively,the therapy might require knowledge of the amino acid sequence (derivedfrom the cDNA sequence), as in the use of reagents that would modifyenzyme activity. The identification of sequences and/or sequence changesin specific regions of the cDNA or protein, such as FAD binding sites orfolate-binding sites, are useful in designing therapeutic protocolsinvolving the above nutrients.

Utility of Invention in Clinical and Diagnostic Studies

Coronary artery disease patients in Montreal (n=153) were studied toexamine the frequency of the alanine to valine substitution. Fourteenpercent of the patients were homozygous for this mutation. An analysisof 70 control individuals (free of cardiovascular disease) demonstratedthat only seven % of these individuals were homozygous for the alanineto valine mutation.

Analysis of homocysteine levels in 123 men of the above patient groupindicated that the mutant allele significantly raised homocysteinelevels from 10.2 micromoles/L in homozygous normal men to 11.5 and 12.7in heterozygotes and homozygous mutants, respectively.

Families with a child with spina bifida, a neural tube defect, have beenexamined for the presence of the alanine to valine mutation.Approximately 16% of mothers who had a child with spina bifida werehomozygous for this mutation, while only 5% of control individuals werehomozygous. Fathers of children with spina bifida also had an increasedprevalence of the homozygous mutant genotype (10%) as did the affectedchildren themselves (13%).

Table 4 indicates the interactive effect of folic acid with thehomozygous mutant alanine to valine change. In a study of families fromFramingham, Mass. and Utah, individuals who were homozygous mutant buthad folate levels above 5 ng/ml did not have increased homocysteinelevels compared to individuals with the normal or heterozygous genotype.However, individuals who were homozygous mutant but had folate levelsbelow 5 ng/ml had homocysteine levels that were significantly higherthan the other genotypes.

TABLE 4 Mean fasting and PML homocysteine levels for different MTHFRgenotypes MTHFR genotype Plasma Normals Heterozygotes HomozygotesHomocysteine (−/−) (+/−) (+/+) P_(trend) N 58 61 30 Fasting* 9.4 9.212.1 0.02 Folate <5 ng/mL 10.2 10.4 15.2 0.002 Folate³ 5 ng/mL 8.2 7.57.5 0.52 Post-Methionine 30.0 30.9 31.3 0.62 load *Significantinteraction between folate levels and genotype (p = 0.03)

Table 4 provides preliminary data for therapeutic intervention by folicacid supplementation to individuals who are homozygous for the alanineto valine change. The data suggest that higher levels of plasma folatewould lead to normalization of homocysteine levels in mutant individualsand might prevent the occurrence of disorders associated with highhomocysteine levels, such as cardiovascular disease, neural tubedefects, and possibly other disorders. Folic acid supplementation formutant individuals might also restore methionine andS-adenosylmethionine levels to normal. This would be relevant fordisorders that are influenced by methylation, such as neoplasias,developmental anomalies, neurologic disease, etc.

Genetic Polymorphism in Methylenetetrahydrofolate Reductase (MTHFR)Associated with Decreased Activity

A common mutation (C677T) results in a thermolabile enzyme with reducedspecific activity (approximately 35% of control values in homozygousmutant individuals). Homozygous mutant individuals (approximately 10% ofNorth Americans) are predisposed to mild hyperhomocysteinemia, whentheir folate status is low. This genetic-nutrient interactive effect isbelieved to increase the risk for neural tube defects (NTD) and vasculardisease. There has been reported an increased risk for spina bifida inchildren with the homozygous mutant genotype for C677T. With the presentinvention, a second common variant in MTHFR (A1298C), an E to Asubstitution has been characterized. Homozygosity was observed inapproximately 10% of Canadian individuals. This polymorphism wasassociated with decreased enzyme activity; homozygotes had approximately60% of control activity in lymphocytes.

A sequence change (C1298A) has been identified. Heterozygotes for boththe C677T and the A1298C mutation, approximately 15% of individuals, had50%-60% of control activity, a value that was lower than that seen insingle heterozygotes for the C677T variant. No individuals werehomozygous for both mutations. A silent genetic variant T1317C, wasidentified in the same exon. It was relatively infrequent (allelefrequency=5%) in the study group, but was common in a small sample ofAfrican individuals (allele frequency=39%).

In addition, by virtue of the role of MTHFR in folate-dependenthomocysteine metabolism, the C677T mutation predisposes to mildhyperhomocysteinemia, a risk factor for vascular disease, in thepresence of low folate status. By the present invention, the frequencyof the A1298C variant has been determined and its potential impact onenzyme function has been assessed.

Patients with spina bifida and mothers of patients were recruited fromthe Spina Bifida Clinic at the Montreal Children's Hospital followingapproval from the Institutional Review Board. Control children andmothers of controls were recruited from the same institution. Bloodsamples were used to prepare DNA from peripheral leukocytes, to assayMTHFR activity in lymphocyte extracts, and to measure total plasmahomocysteine (tHcy). The presence of the C677T mutation (A to V) wasevaluated by PCR and HinfI digestion (2). The A1298C mutation wasinitially examined by PCR and MboII digestion (5). The silent mutation,T1317C, was identified by SSCP and sequence analysis in a patient withsevere MTHFR deficiency and homocystinuria. This patient, anAfrican-American female, already carries a previously-described splicemutation (patient 354 (8)). Since this mutation also creates a MboIIsite and results in a digestion pattern identical to that of the A1298Cmutation, distinct artificially-created restriction sites were used todistinguish between these 2 mutations. Detection of the A1298Cpolymorphism was performed with the use of the sense primer5′-GGGAGGAGCTGACCAGTGCAG-3′ and the antisense primer(5′-GGGGTCAGGCCAGGGGCAG-3′), such that the 138bp PCR fragment wasdigested into 119bp and 19bp fragments by Fnu4HI in the presence of theC allele. An antisense primer (5′-GGTTCTCCCGAGAGGTAAAGATC-3′), whichintroduces a TaqI site, was similarly designed to identify the C alleleof the T1317C polymorphism. Together with a sense primer(5′-CTGGGGATGTGGTGGCACTGC-3′), the 227bp fragment is digested into 202bpand 25bp fragments.

TABLE 5 Genotype distributions, MTHFR activity (nmol formaldehyde/mgprotein/hour), and total plasma homocysteine (tHcy; μM) for mothers andchildren E/E E/A A/A A/A A/V V/V A/A A/V V/V A/A A/V V/V Mothers (n =141) # 24 32 19 27 26 0 13 0 0 % 17 23 13 19 18 0  9 0 0 MTHFR 49.0 ±18.9 33.0 ± 10.8 15.7 ± 4.5 45.0 ± 16.0 30.2 ± 19.3 — 32.1 ± 9.0  — —[14]  [19]*  [11]* [15]  [15]*  [7]* THcy 9.5 ± 3.1 10.0 ± 3.2  12.2 ±7.1 8.4 ± 2.1 10.0 ± 3.1  — 9.5 ± 2.0 — — [24] [32]  [19]** [25] [26][13] Children (n = 133) # 23 43 18 20 15 1 13 0 0 % 17 32 13 15 11 1 100 0 MTHFR 52.0 ± 17.0 38.2 ± 15.0 16.2 ± 5.3 35.7 ± 9.7  26.1 ± 5.0  21.6 29.5 ± 10.3 — — [12]  [27]*  [11]*  [18]*  [9]* [1]  [6]* Thcy 7.6± 2.5 8.2 ± 3.0  9.7 ± 5.1 7.5 ± 2.3 8.1 ± 2.8   9.5 7.4 ± 1.5 — — [23][43]  [18]** [20] [15] [1] [13] The three A1298C genotypes and the threeC677T genotypes are designated by the amino acid codes: EE, EA, AA, andAA, AV, VV, respectively. Statistical significance was assessed bystudent t-test, in comparison with EEAA values. *(p < 0.05); **(p ≦0.07). Standard deviations are given and square brackets indicate thenumber of individuals for whom MTHFR activities and homocysteine levelswere available.

The frequencies of the three genotypes for the A1298C mutation (EE, EAand AA) were not different between case and control mothers, or betweencase and control children (data not shown). Consequently, all themothers and all the children were grouped together for analyses (Table5). Nine % of mothers had the homozygous AA genotype while 37% wereheterozygous. This frequency is quite similar to the frequency of thehomozygous mutant genotype (VV) for the C677T polymorphism. In the MTHFRhuman cDNA sequence mentioned above, the cDNA contained the C nucleotideat bp 1298 change as a C1298A substitution. Since the A nucleotide isclearly the more frequent base at this position, the A1298C nomenclaturewas chosen.

Since the C677T mutation (A to V) decreases MTHFR activity and increaseshomocysteine levels, the three genotype groups for the A1298C (E to A)mutation were further stratified by the genotype for the A to Vmutation, to avoid the confounding influence of the latter polymorphismon MTHFR activity and homocysteine levels. The frequencies of the 9genotypes, with MTHFR activity and homocysteine levels for eachgenotype, are shown in Table 5. If the mothers and children withouteither mutation i.e. EE/AA are used as the reference (control) group,the mothers and children that are homozygous for the A1298C mutation(AAAA) have approximately 65% and 57%, respectively, of control MTHFRactivity. Heterozygotes for the C677T change alone (EEAV) haveapproximately 70% of control activity, as reported in other studies,while double heterozygotes (EAAV), 18% of mothers and 11% of children,have an additional loss of activity (approximately 62% and 50% ofcontrol values, respectively).

Homocysteine levels were not significantly increased by the A1298Cmutation, but homocysteine was elevated (with borderline significance,p≦0.07) in mothers and children who were homozygous for the C677Tchange. The small number of individuals who were homozygous for theA1298C mutation (n=13) may have influenced the power of the statisticalanalyses and precluded an investigation of the genetic-nutrientinteractive effect that leads to mild hyperhomocysteinemia, as seen inindividuals with the C677T mutation.

The T1317C substitution does not alter the amino acid (phenylalanine)and is likely a benign change, although a splicing defect cannot beruled out at the present time. In an evaluation of 38 control mothersfrom this study, 2 were found to be heterozygous and one was identifiedas a homozygote, resulting in an allele frequency of 5% ( 4/76). Sincethis substitution was identified in an African-American female, controlAfrican individuals were also examined (n=9). Seven of these wereheterozygous, resulting in an allele frequency of 39% ( 7/18).

The A1298C mutation clearly reduces MTHFR activity, albeit to a lesserextent than the C677T mutation. Consequently its effect on homocysteinelevels is also attenuated and, in fact, may only be significant when anindividual carries both mutations and/or has poor nutrient status.However, since double heterozygotes are estimated to representapproximately 15% of the population, this variant should be examined inconjunction with the C677T variant in studies of hyperhomocysteinemia.

The A1298C mutation is clearly polymorphic in Canadian individuals andshould be examined in other populations. The A nucleotide is likely tobe the ancestral sequence since it represents the more common allele,although the original human MTHFR cDNA sequence (GenBank accessionnumber U09806) carried the C nucleotide. This polymorphism is similar infrequency to the C677T polymorphism. Presumably the two substitutionsarose separately on a A1298/C677 or E/A haplotype, since the haplotypewith both substitutions (C1298/T677 or A/V) is extremely rare. One suchhaplotype was seen in a child with the EAVV genotype, suggesting arecombinant chromosome.

Doubly homozygous individuals (AAVV) were not observed in this study.Since the double mutation in cis is rare, it is possible that not enoughalleles were studied. Larger studies in other populations might resultin the identification of these individuals. Presumably the MTHFRactivity would be even lower and homocysteine levels might be higherthan those observed thus far.

The C677T polymorphism in exon 4 is within the N-terminal catalyticdomain of the enzyme whereas the A1298C polymorphism in exon 7 is withinthe C-terminal regulatory domain. The more dramatic effect on enzymeactivity with the first polymorphism may be a consequence of itslocation within the catalytic region. The second polymorphism couldaffect enzyme regulation, possibly by S-adenosylmethionine, anallosteric inhibitor of MTHFR, which is known to bind in the C-terminalregion.

Many studies have examined the effects of the C677T polymorphism onMTHFR enzyme activity and on homocysteine levels. Although thecorrelation between the presence of this substitution and decreasedenzyme activity/increased homocysteine levels has been quite good, thevariability in results, particularly in heterozygous individuals, mayreflect the presence of a second common variant in the population.

The third variant, T1317C, was present on 5% of alleles in Canadianindividuals but appears to be extremely common in individuals of Africanancestry. The methodology outlined in this report should be used toassess the frequency of the A1298C and T1317C in other populations,since the use of the MboII restriction site for analysis of the A1298Cchange, as first reported, would not discriminate between the 2polymorphisms.

The C677T mutation is a risk factor for hyperhomocysteinemia and hasbeen implicated in both neural tube defects and vascular disease.

Gene Structure of Human and Mouse Methylenetetrahydrofolate Reductase(MTHFR)

A human cDNA for MTHFR, 2.2 kb in length, has been expressed and shownto result in a catalytically-active enzyme of approximately 70 kDa.Fifteen mutations have been identified in the MTHFR gene: 14 raremutations associated with severe enzymatic deficiency and one commonvariant associated with a milder deficiency. The common polymorphism hasbeen implicated in three multifactorial diseases: occlusive vasculardisease, neural tube defects and colon cancer. The human gene has beenmapped to chromosomal region 1p36.3 while the mouse gene has beenlocalized to distal Chromosome 4. The isolation and characterization ofthe human and mouse genes for MTHFR is herein reported. A human genomicclone (17 kb) was found to contain the entire cDNA sequence of 2.2 kb;there were 11 exons ranging in size from 102 bp to 432 bp. Intron sizesranged from 250 bp to 1.5 kb with one exception of 4.2 kb. The mousegenomic clones (19 kb) start 7 kb 5′ to exon 1 and extend to the end ofthe coding sequence. The mouse amino acid sequence is approximately 90%identical to the corresponding human sequence. The exon sizes, locationsof intronic boundaries, and intron sizes are also quite similar betweenthe two species. The availability of human genomic clones has beenuseful in designing primers for exon amplification and mutationdetection. The mouse genomic clones may be used to make constructs forgene targeting and generation of mouse models for MTHFR deficiency.

A common polymorphism, C677T has been identified, which converts analanine codon to valine (Frosst et al., 1995). This common polymorphism,which is present on approximately 35% of alleles in the North Americanpopulation, encodes the thermolabile variant and predisposes to mildhyperhomocysteinemia when folate status is low (Frosst et al., 1995;Jacques et al., 1996; Christensen et al., 1997). This genetic-nutrientinteractive effect is believed to be a risk factor for arteriosclerosis(Frosst et al, 1995) and neural tube defects. In contrast, the mutanthomozygous genotype may decrease the risk for colon cancer.

The characterization of the genomic structure for human MTHFR isreported herein. The corresponding analysis of the mouse gene, with acomparison of the overall organization of the gene and the amino acidsequences in these two species, is also shown.

Screening of Genomic Libraries

Genomic libraries were screened using standard methods of plaquehybridization. The 2.2 kb human cDNA was radiolabelled and used as aprobe in screening both human and murine genomic libraries. Screeningfor the human gene was performed on a phage library of partial EcoRIdigestion fragments from total genomic DNA (ATCC #37385), and on a phagelibrary of chromosome 1-specific complete EcoRI digestion fragments(ATCC #57738). Screening for the mouse gene was performed on a λDASHlibrary of partial Sau3A digestion fragments from total genomic DNA ofmouse strain 129SV (obtained from Dr. J. Rossant, University ofToronto). Positive clones were purified by sequential rounds ofscreening and isolation, and phage DNA was isolated using phage DNAisolation columns (QIAGEN). Human clones were digested with EcoRI torelease the inserts, and then with XbaI to facilitate cloning intoBluescript plasmid (Stratagene). The mouse clones were digested withSalI or EcoRI, and the inserts were subcloned into Bluescript.

Characterization of Mouse cDNA Sequences

Mouse genomic clones were sequenced (Sequenase kit, Amersham) usinghuman cDNA primers spanning most of the available 2.2 kb cDNA. Thesesequences were then used to generate mouse-specific cDNA primers. Themouse-specific primers were used in PCR amplification of overlappingcDNA fragments from reverse-transcribed mouse liver RNA. The PCRproducts were subcloned into the PCRII vector (Invitrogen) andsequenced. Two different species of mouse (C57B1/6J and ct) were used togenerate MTHFR sequence by RT-PCR, to ensure that the PCR protocol didnot generate sequencing errors.

Characterization of Intron Boundaries and Sizes, and RestrictionAnalysis of Human and Mouse Genes

Primers from cDNA sequences of human and mouse were used to sequence therespective genomic clones. Intron boundaries were determined fromregions of divergence between cDNA and genomic clone sequences, and bythe identification of splice acceptor and donor consensus sites.Intronic sequences were obtained for 40-50 bp from the junctions and areshown in FIGS. 12A-12B (human) and FIGS. 13A-13B (mouse). The same cDNAprimers were used in PCR amplification of total genomic DNA and ofgenomic clones to determine the approximate sizes of introns in thehuman and mouse genes. Table 6 lists the locations and approximate sizesof introns for both species. The PCR products were analyzed byrestriction enzyme digestion to generate a preliminary restriction mapof the gene. This restriction map was then confirmed by restrictionanalysis of the genomic clones in Bluescript.

Referring to FIGS. 12A-12B, the bp location of the exons within thecDNA, in parentheses, is based on the published human cDNA sequence(GenBank accession number U09806). Bp 1 is 12 bp upstream from the ATGin the original cDNA; an asterisk indicates the equivalent base here.Exon 1 contains the ATG start site (underlined), and exon 11 containsthe termination codon (underlined). Uppercase characters indicate exonicsequences, and lower case characters are intronic. Consensus splicejunction sequences are underlined. The 3′ boundary of exon 11 has beendesignated by the location of the polyA tail.

Referring to FIGS. 13A-13B, the bp location of the exons within thecDNA, in parentheses, is based on the equivalent bp 1 of the humansequences in FIGS. 12A-12B (bp 1 is indicated by an asterisk). Exon 1contains the ATG start site (underlined), and exon 11 contains thetermination codon (doubly underlined). Uppercase characters indicateexonic sequences, and lower case characters are intronic. Consensussplice junction sequences are underlined. The 3′ boundary of exon 11 isdesignated as the termination codon, since the site of polyadenylationis unknown. Also underlined in exon 11 is the first repeat of the 52 bprepeated element.

Referring to FIG. 14, exon sizes for human and mouse are reported inFIGS. 12A-12B and FIGS. 13A-13B, respectively. Exons are indicated inshaded boxes. Uncharacterized regions of the gene are hatched, and exonnumbering corresponds to FIGS. 12A-12B and 13A-13B. E=EcoRI; X=XbaI;A_(n)=polyadenylation site. The EcoRI restriction site at the 5′ end ofthe mouse gene is part of the phage polylinker sequence.

Referring to FIG. 15, residues that are identical to the human MTHFRsequence are shown as empty boxes, and gaps in amino acid homology arerepresented by a dash.

Human Genomic Clones

The genomic clones isolated from the human libraries contained a 16 kbEcoRI fragment, encompassing part of exon 1 and exons 2 through 11, anda 1 kb EcoRI fragment containing most of exon 1. Exon 1 is defined asthe most 5′ exon from the previously published cDNA sequence; itcontains the ATG start site that was used to express the human cDNA inbacterial extracts (Frosst et al. 1995). A graphic representation of thehuman gene and its restriction map are depicted in FIG. 3. The sequencesof each exon and 50 bp of flanking intronic sequences are shown in FIGS.12A-12B. Exons range in size from 102 bp to 432 bp, and the criticaldinucleotides in the 5′ and 3′ splice site consensus sequences (GT andAG, respectively) are underlined. The 3′ boundary of exon 11 is definedby the site of polyadenylation in the cDNA; a possible polyadenylationsignal (AACCTA) is present 15 bp upstream of the polyadenylation site,although it varies from the consensus sequence. Table 6 lists thelocations and approximate sizes of introns as determined by PCRamplification; the introns range in size from approximately 250 bp to4.2 kb.

Mouse Genomic Clones

Genomic clones isolated from the mouse libraries were digested withEcoRI for subcloning and characterization. Exon nomenclature is based onthe corresponding human gene sequences. FIGS. 13A-13B list all knownexons and their sizes, with 40 to 50 bp of flanking intronic sequences.FIG. 14 shows a graphic representation of the mouse genomic structurealigned with the human gene. The size of exon 11 is undetermined, sincethe sequence for this region was determined directly from the genomicclones. The termination codon is located within a region of 52 bp whichis repeated 3 times in the gene. The significance of this, if any, isunknown at the present time. The dinucleotides of the splice junctions(underlined in FIGS. 13A-13B) are in agreement with consensus sequences.Table 6 lists the approximate sizes of introns as determined by PCR, andtheir bp location in the cDNA. The introns range in size fromapproximately 250 bp to 4.2 kb.

Comparison of the Human and Mouse Genes

The human and mouse genes are very similar in size and structure (FIG.14). The introns are similar in size, and identical in location withinthe coding sequence. However, the mouse cDNA is one amino acid shorterin exon 1 which causes a shift in bp numbering of the mouse cDNA (Table6, FIGS. 13A-13B). Exon 1 was defined from the original published humancDNA, based on the presence of a translation start codon. In both humanand mouse genes, the 5′ boundary of exon 1 was assigned after theisolation of several non-coding cDNA extensions that are generated byalternative splicing from this junction. Characterization of these 5′cDNA extensions is in progress. The nucleotide sequences of the humanand mouse genes are very similar within coding regions, but homologydecreases dramatically in the 3′ UTR region and within introns.

Human and Mouse Primary Amino Acid Sequence Homology

The primary amino acid sequences of human and mouse were compared toeach other, and aligned with the sequence of the metF (MTHFR) enzymefrom bacteria (FIG. 15). The human and mouse amino acid sequences arealmost 90% identical. As previously observed, only the 5′ half of themammalian sequences align with the bacterial enzyme; bacterial MTHFR hasthe same catalytic activity as the mammalian enzyme but lacks theregulatory region in the C-terminal domain. The murine amino acidsequence is two amino acids shorter than the human sequence: one lessamino acid in exon 1 and one less in exon 11.

The isolation of the human MTHFR gene and the analysis of gene structureare part of an ongoing effort to study MTHFR deficiency inhomocystinuria and in multifactorial diseases. The availability ofgenetic structure information and of intronic sequences will help in themutational analysis of patients suffering from MTHFR deficiency and inthe characterization of the 5′ regulatory region.

Expression analysis of the 2.2 kb cDNA in a bacterial expression systemresulted in a catalytically-active 70 kDa protein (Frosst et al. 1995).A MTHFR polypeptide of this size was observed in some human tissues onWestern blots, but a larger isozyme (77 kDa), corresponding to theestimated size of the porcine polypeptide, was observed in all theexamined tissues. These data suggested the presence of protein isoformsfor MTHFR that could be tissue-specific (Frosst et al., 1995). Sincehuman or mouse sequences homologous to the N-terminal porcine amino acidsequences have not been identified, it is assumed that the missingsequences required to encode the larger isoform are 5′ to the availablecDNA sequences. Two mRNAs for human MTHFR (approximately 7.5 and 8.5 kb)have been seen in all tissues on Northern blots (data not shown),suggesting very large UTRs. The isolation of 5′ coding sequences hasbeen complicated by the presence of several alternatively-spliced 5′non-coding extensions that splice into exon 1. The alternative splicinginto exon 1 has been observed in both human and mouse MTHFR. The longUTRs and the alternative splicing events suggest that the regulation ofthis important gene may be quite complex.

Nonetheless, the available information has been critical foridentification of mutations in patients with various forms of MTHFRdeficiency. The mouse sequences in exons 1 and 2 have been useful in thedesign of antisense oligonucleotides to successfully inhibit MTHFR inmouse embryo cultures and disrupt development of the neural tube (Lanoueet al. 1997). The isolation and characterization of mouse genomic clonesis essential for construction of targeting vectors to generate mousemodels for MTHFR deficiency.

TABLE 6 Approximate sizes of introns, and their locations in human andmouse MTHFR cDNA Approximate Human Mouse Intron size (kb) location¹location¹ 1 1.5 248-249 245-246 2 0.8 487-488 484-485 3 4.2 598-599595-596 4 0.8 792-793 789-790 5 0.35 1043-1044 1040-1041 6 0.251178-1179 1175-1176 7 0.3 1359-1360 1356-1357 8 1.5 1542-1543 1539-15409 1.3 1644-1645 1641-1642 10 0.3 1764-1765 1761-1762 ¹Base pairsflanking introns, from FIGS. 1 and 2. Bp1 is 12 bp upstream from theATG, as in the original report of the cDNA sequence (Goyette et al.1994).

Various doses of methotrexate (a drug used in treatment of cancer andarthritis, possibly other diseases) were added to colon carcinoma linesin culture. Much lower doses of methotrexate are needed to kill thelines that carry the 677C/T mutation in MTHFR, compared to lines that donot carry this mutation. The IC50 (concentration needed to kill half thecells) is approximately 20 nM for lines with the mutation andapproximately 150 nM for lines without the mutation. To extrapolate tothe human condition, patients with this MTHFR mutation might requirelower doses of methotrexate for therapy, or might be subject tomethotrexate toxicity at high doses.

TABLE 7 Summary of BMD analysis Entire cohort: Values are means +/− SEGenotype 1/1 Genotype 1/2 Genotype 2/2 Spinal Z score −1.06 (0.193)−1.25 (0.176) −1.86 (0.238) Femoral neck Z score −0.69 (0.134) −0.78(0.126) −0.87 (0.228) Trochanter Z score −0.60 (0.142) −0.52 (0.134)−1.15 (0.249) Ward's triangle Z −0.67 (0.147) −0.80 (0.139) −0.96(0.257) score Table 7 indicates the values for bone mineral density in agroup of individuals who were examined for the MTHFR C677T mutation.Genotype 1/1 = normal C/C Genotype 1/2 = camers C/T Genotype 2/2 =homozygons mutant T/T

As seen on Table 7, the lower the score, the lower the bone mineraldensity and therefore the higher the risk for osteoporosis. The resultssuggest that the homozygons mutant genotype (2/2) is associated withlower bone mineral density and therefore higher risk of osteoporosis.

Diagnosis and Therapeutic Outcome of a Psychosis

MTHFR alleles are also useful as diagnostic, therapeutic, prognostic,and pharmacogenomic markers for neurological disorders such as psychosesand in other diseases and disorders that may be treated withanti-psychotic therapeutics.

Psychoses are serious, debilitating mental illnesses which arecharacterized by loss of contact with reality and may also include anyof the following symptoms: delusions, hallucinations, thought disorder,strange behavior or emotions, or withdrawal from social contact. Theduration of untreated psychosis may be correlated with worsenedpatient-prognosis.

Schizophrenia is the most common psychosis, which affects approximately2 million Americans. Although genetic factors are known to play a rolein the etiology of schizophrenia, identification of susceptibility geneshas been difficult. This difficulty may be due partially to thevariability of the schizophrenia phenotype, including variations inseverity of symptoms, response to medications, and long-term outcome.Indeed, one third to one quarter of schizophrenic subjects remainseverely symptomatic despite multiple trials with individual orcombinations of anti-psychotic therapeutics, also called neuroleptics.This variability may reflect factors such as genetic heterogeneity orthe presence of modifier genes. Compared to neuroleptic nonresponders,the group of responders is characterized by a high female to male ratio,better long-term outcome, and more frequently disturbed indices ofdopamine neurotransmission.

In addition to the inability to reduce symptoms in a substantial numberof schizophrenia patients, current neuroleptic therapeutics also cause avariety of serious side effects. Examples of the adverse effects ofconventional neuroleptics include cardiac conduction abnormalities canresult in patient deaths, seizures, involuntary repetitive movements(tardive dyskinesia), dulling of cognition, sedation, weight gain,sexual or reproductive dysfunction, blood dyscrasias, jaundice, skinreactions such as uticaria and dermatitis, and extrapyramdial effects.Extrapyramdial side effects involve conditions such as abnormal facialexpressions, restlessness or constant movement, and rigidity and tremorat rest (parkinsonian syndrome). Because of the severity of these sideeffects and the low therapeutic-to-toxic index of conventionalneuroleptics, other neuroleptics, called atypical neuroleptics, havebeen recently developed. Atypical neuroleptics have a lower incidence ofextrapyramidal symptoms and tardive dyskinesia; however, they are stillassociated with weight gain and effects on blood pressure and liverfunction, as observed for conventional neuroleptics.

Due to the severity and heterogeneity of schizophrenia, improved methodsare needed for diagnosing people at risk for or suffering fromschizophrenia. Improved methods are also desirable for determining apreferred therapy for a particular diagnosed individual or group ofindividuals. The selection of a preferred therapy for a particularsubject may prevent the subject from having to endure possiblyirreversible side effects from therapies that are ineffective for thatsubject. Reduced side effects of the preferred therapy compared to othertherapies may result in greater patient compliance, further increasingthe likelihood of therapeutic benefit from the therapy.

We have discovered that the heterozygous form of the C677T mutation inmethylenetetrahydrofolate reductase (MTHFR) is more common in patientsdiagnosed with schizophrenia than in a healthy control population andthus is a risk factor for this disease. In addition, both theheterozygous and homozygous forms of this mutation are associated withan improved response to neuroleptic treatment and long-term outcome.

Because the C677T mutation results in decreased MTHFR activity andincreased enzyme thermolability, other MTHFR genotypes or haplotypeshaving similar characteristics may be associated with schizophrenia orimproved response to neuroleptics. For example, another mutation ofhuman MTHFR, A1298C which converts glutamic acid to alanine, alsoresults in reduced activity in the encoded protein, and individuals whoare heterozygous for both the C677T and A1298C mutations have even lowerMTHFR activity and have higher homocysteine levels than observed inindividuals with the single C677T mutation. Other similar mutationsinclude, but are not limited to, G167A, G482A, C559T, C692T, C764T, aG/A G792+1A, C985T, C1015T, C1081T, T1317C, and any combination of theseMTHFR mutations.

In this study, two groups of schizophrenic patients selected on thebasis of a priori defined criteria of long-term outcome and response toneuroleptics (excellent responders and very poor responders, Table 8)and a control group of health volunteers were compared with regard tothe C-to-T substitution at nucleotide 677 of the MTHFR gene, called “Vallele.”

TABLE 8 Demographic and clinical characteristics of patients andcontrols Nonresponders Responders (n = 62) (n = 43) Age (yr.) ± SD 38.7± 6.9 40.6 ± 10.5  Gender (% Males) 74 67 Age at 1 C (yr.) 18.1 ± 3.924.2 ± 4.8** Subtype, U/P/D/C 26/30/4/1 6/36/1/0** Time as in-patient(%) 63.5 ± 38   8.2 ± 11.8** BPRS score 48.9 ± 9.0 24.4 ± 3.9** Age at 1C = age at first contact with a psychiatric care facility, U =undifferentiated, P = paranoid, D = disorganized, C = catatonic, BPRS =Brief psychiatric rating scale.

The heterozygous and homozygous C677T mutations were found morefrequently in schizophrenia patients than in control subjects (Table 9).In addition, there was a significant association between schizophreniaand the presence of at least one C677T allele of the MTHFR gene(χ²=5.44, degrees of freedom (df)=1, p=0.019). This association was dueto an over-representation of allele V in neuroleptic responder patientscompared to controls (χ²=16.77, df=1, p=0.00004); non-responder patientsdid not differ from controls. In addition, the attributable fractionassociated with carrying at least one V allele in the group ofneuroleptic responder patients was

TABLE 9 Analysis of genotypic and allelic association of 677C→Tpolymorphism in MTHFR gene. N of subjects with genotype (%) χ² statisticC vs. R Number of alleles (%) χ² statistic A/A V/A V/V (df = 1). A V (df= 1) All subjects C 41 (44.5) 36 (40.0) 13 (14.4)  D: χ² = 16.12, 117(65.0)  63 (35.0) C vs. R: χ² = 17.52, p = 0.00003 R  5 (11.6) 23 (53.5)15 (34.9)  p = 0.00006 33 (38.4) 53 (61.6) C vs, NR: χ² = 0.10, p = 0.74NR 25 (40.3) 29 (46.8) 8 (12.9) R: χ² = 7.49, p = 0.006 79 (63.7) 45(36.3) Males C 17 (41.4) 18 (43.9) 6 (14.7) D: χ² = 14.0, p = 0.0001 53(63.4) 30 (36.6) χ² = 12.77, p = 0.00035 R 1 (3.5) 17 (58.6) 11 (38.8) R: χ² = 5.01, p = 0.025 19 (32.7) 39 (67.3) Females C 24 (49.0) 18(36.7) 7 (14.3) D: χ² = 2.02, p = 0.15 66 (67.3) 32 (32.7) χ² = 2.82, p= 0.092 R  4 (28.6)  6 (42.9) 4 (28.5) R: χ² = 1.63, p = 0.20 14 (50.0)14 (50.0) D = dominant model, R = recessive model, C = controls, R = NLPresponder patients, NR = NLP non responder patients. P-values are notcorrected for multiples testing75%, meaning that 75% of the schizophrenia cases may be due to thismutation. The association between responder patients and the C677Tmutation was statistically stronger in males than in females.

The association between the C677T mutation and schizophrenia wasobserved for both a recessive (VV vs. AV+AA) as well as a dominant model(VV+AV vs. AA) of transmission of this mutation; however, the lattershowed consistently more power to discriminate between responsivepatients and controls (Table 9).

These results indicate that good response to neuroleptic medication andgood long-term outcome characterize the schizophrenia phenotypeassociated with allele V of the MTHFR gene. The fact that no associationwith the V allele was observed in the groups of neuroleptic nonresponderpatients indicates, as previously suggested, that these two groups ofpatients are at least partially different from a pathogenic point ofview. The dosage effect of allele V on age-at-onset, a marker ofseverity of illness that is independent from responsiveness toneuroleptics, suggests that this association is possibly mediated by aschizophrenia subtype. Thus, the classification of schizophrenicpatients by the presence or absence of the C677T mutation may be aneffective strategy to reduce heterogeneity for studies of the treatmentor biology of schizophrenia. Additionally, the association of allele Vwith the ability of neuroleptic treatment to reverse psychotic symptomsand possibly the deterioration associated with untreated psychosis maybe due to the association of allele V with an ability to respond toneuroleptics that is independent of schizophrenia subtype.

While not meant to limit the invention to a particular theory, it alsopossible the V allele is associated with a disturbance in the one-carboncycle metabolism regulated by MTHFR, since MTHFR provides the carbongroup for the generation of S-adenosylmethionine, a universal methyldonor. A disturbance of one-carbon metabolism may result in a deficiencyof methylation reactions that are required for numerous pathwaysincluding detoxification, DNA methylation and regulation of biogenicamine metabolism. Another possibility is that the V allelic variant mayact as a moderator of the severity of schizophrenia by increasing theplasma concentration of homocysteine. Indeed it has been reported that ahomocysteine load causes worsening of psychotic symptoms inschizophrenic patients.

The association of other MTHFR alleles with schizophrenia, or any otherdisease, or with response to particular therapies may be determined byany standard methods (see, for example, those methods described in WO00/04194; Ueda et al., Circulation 98:2148-2153, 1998, Tan et al.,Lancet 350:995-999, 1997; Poirier et al., Proc. Natl. Acad. Sci. USA92:12260-12264, 1995; Spire-Vayron de la Moureyre et al., British J. ofPharmacology 125:879-887, 1998; Drazen et al., Nature Genetics22:168-170, 1999; Smeraldi et al., Molecular Psychiatry 3:508-511, 1998;Kuivenhoven et al. New Eng. J. of Med. 338(2):86-93, 1998; Aarranz etal., Molecular Psychiatry 3:61-66, 1998). Haplotyping may also be usedto determine associations between particular mutations or combination ofmutations and particular diseases or responses to particular therapies.

Clinical trials for an anti-psychotic therapy may be conducted asdescribed below or using any other appropriate method. The subjects inthe trial may be stratified based upon their MTHFR genotype at therelevant polymorphic sites. This stratification may be used to correlatean MTHFR genotype or haplotype with the efficacy or the safety of thetherapy and may be performed before, during, or after the clinicaltrial. The correlation of the safety and efficacy of the therapy insubjects with a particular MTHFR mutation or combination of MTHFRmutations may be considered during the Food and Drug Administration(FDA) approval process and used to determine preferred therapies for thedifferent MTHFR subgroups of subjects.

Briefly, clinical trials generally consist of four phases: Phase I is asafety study generally involving healthy volunteers; Phase II is adose-ranging, efficacy study involving subjects diagnosed with acondition, and Phase III is a larger safety and efficacy study. If theefficacy data from the Phase III trial are statistically significant,the therapy may be approved by the FDA. Phase IV is a post-markingapproval trial that may be used to further refine the approvedindication population or to compare one therapy to another one. Thisprocess for testing a therapy in humans may also be modified by oneskilled in the art depending on specific pharmacokinetic orpharmacodynamic properties of the therapy or the availability ofsubjects diagnosed with the condition. For example, cytotoxic therapies,such as cancer chemotherapeutics, are not tested in healthy controlsubjects and generally begin clinical trials with a Phase 11 study.

Clinical trials may be performed using standard protocols (see, forexample, Spilker, Guide to Clinical Trials, Raven Press, 1991; Tygstrup,ed., The Randomized Clinical Trial and Therapeutic Decisions MarcelDekker; Thall, ed. Recent Advances in Clinical Trial Design andAnalysis, Cancer Treatment and Research, Ctar 75, Kluwer Academic, 1995;and Piantadosi, Clinical Trials: A Methodologic Perspective, WileySeries in Probability and Statistics, 1997). Examples of possibleclinical trials protocols include placebo-controlled, single blinded,double blinded, crossover, and other studies.

For the design of clinical trials to test the contribution of an MTHFRmutation or a combination of MTHFR mutations to variation in patientresponse to a therapy, one skilled in the art may use readily availablemethods to determine the appropriate parameters for a particularclinical trial. Examples of such factors include the genetic hypothesisto be tested, the number of subjects required to observe a statisticallysignificant effect (power analysis), the appropriate dosing range, theduration of the trial, and the appropriate endpoints to measure theresponse to the therapy. The applications of genetic hypothesis testing,power analysis, and statistical analysis of patient response to eachphase of a clinical trial have been described previously. Additionally,haplotyping may be used to detect which MTHFR genotype at which positionmay be correlated with a particular response to a particular therapy.

The endpoints of a clinical trial of an anti-psychotic therapy mayinclude factors associated with the conditions. Examples of possibleendpoints include semi-objective grading scales administered by clinicalstaff. Several standard grading scales have been developed and acceptedas assessment tools of the efficacy of a therapy in clinical trials andinclude, but are not limited to, cognitive assessment, pain, psychotic,behavioral, and global clinical impression scales.

A further consideration for the design of a clinical trial for acompound in the category of psychiatric illness is the duration of thetrial. It may take weeks to months before a “wash-out” can be achieved(i.e. reduction of the dose to the point of removal of therapy) for thetherapies that the subjects had been receiving prior to the clinicaltrial. Additionally, many anti-psychotics exhibit dose-dependentefficacy and often require 2-3 weeks before empirical clinical globalimpression and semi-objective grading scale analysis suggests atherapeutic dose has been achieved. Both the “run-in” period for theachievement of a therapeutic dose and the “run-out” period for thesubsequent decrease in the dose for removal of the therapy after theclinical trial must be performed under controlled conditions.

The present invention provides a number of advantages. For example, themethods described herein allow for the determination of a subject atrisk for a psychosis for the timely administration of a prophylactictherapy to prevent or delay the psychosis. Thus, this determination ofincreased risk for a psychosis may occur before the presence ofdebilitating symptoms that might otherwise be required for a definitivediagnosis. The methods of the invention also have the advantage of onlyrequiring the presence of one mutation in one MTHFR allele instead ofrequiring a homozygous mutation for the association of the mutation witha psychosis; this is a particular advantage in the diagnosis ofschizophrenia which was previously correlated exclusively withhomozygous patient genotypes. Additionally, the presence of mutations inMTHFR alleles may be easily and rapidly determined using standardtechniques. Moreover, the administration of a preferred therapy tosubject at risk for or diagnosed with from a psychosis may result inimproved patient-outcome, improved drug-efficacy, or reduceddrug-toxicity compared to the administration of other therapies. Theseresults may reduce healthcare costs by decreasing the number of requireddoctor visits or hospitalizations.

The following example is provided to illustrate the invention. It is notmeant to limit the invention in any way.

Association of the C677T MTHFR Mutation with Schizophrenia, Response toTherapy, and Long-Term Outcome Selection of Patients

Nonresponding schizophrenic patients (NR, n=43) were selected from alist of schizophrenic patients identified as candidates for treatment ortreated with atypical neuroleptics because of treatment resistance toconventional neuropleptics. Three institutions provided these NRpatients: Douglas Hospital, Clinique Jeunes Adultes of L.H. LafontaineHospital, and the Schizophrenia Clinic of the Royal Ottawa Hospital.Responding patients (R, n=62) were selected from a list of all patientswho were considered very good responders to conventional neuroleptics bytheir treating physician and/or nurse and who were followed in theoutpatient clinics attached to the Douglas and L.H. Lafontainehospitals, which are the only two university-affiliated psychiatrichospitals for the Montreal intra-muros region.

The following criteria for response or resistance to conventionalneuroleptics were used in the selection of NR schizophrenic patients.None of the NR patients had experienced remission of psychotic symptomswithin the past two years. In the preceding five years, all NR patientshad undergone at least three periods of treatment with conventionalneuroleptics from at least two distinct families of drugs at a doseequal to, or greater than, 750 mg chlorpromazine (CPZ) equivalents formonotherapy or 1000 mg CPZ for therapy with a combination ofneuroleptics. The treatments, which lasted for a continuous period of atleast 6 weeks, resulted in no significant decrease in symptoms. Finally,all patients were unable to function without supervision in all, ornearly all, domains of social and vocational activities and had a GlobalAssessment Score (GAS) (Endicott et al., Arch. Gen. Psychiatry33:766-771, 1976) greater than 40 within the last 12 months. Decisionfor non-response was established by the research psychiatrist uponreview of medical records, with particular attention to: (i) unusualthought content, (ii) hallucinations, (iii) conceptual disorganization,(iv) motor retardation, and (v) emotional withdrawal (items 3, 4, 12, 13and 15 of the Brief Psychiatric Rating Scale; BPRS) (Woerner et al.,Psychopharmacol. Bull. 24:112-117, 1988). At the time of enrollment, aminimal score of 4 (moderate to severe) on at least three of the fiveBPRS items (3, 4, 12, 13, 15), a total BPRS score of at least 45 and/ora CGI (Clinical global Impression) score of at least 5 (markedly ill)were required. These criteria for treatment resistance were derived fromthose of Kane et al. (Arch. Gen. Psychiatry 45(9):789-96, 1988).

Among the selected R patients, all patients had been admitted at leastonce to a psychiatric institution because of an acute psychotic episode.During each hospitalization, patients experienced a full or partialremission in response to treatment with conventional neuroleptics within6 to 8 weeks of continuous treatment. All patients were able to functionautonomously with only occasional supervision in all, or nearly all,domains of social and vocational activities. None of the R patients hadto be admitted to hospitals because of psychotic exacerbation whileunder continuous neuroleptic treatment. All R patients had at least onepsychotic relapse when neuroleptic medication was reduced ordiscontinued. Remission was defined as a complete or quasi-completedisappearance of schizophrenic symptoms, with limited residual symptoms,based on the treating psychiatrist clinical evaluation and hospitalrecords. At the time of enrollment, total BPRS scores were less than 30with no more than one item scoring 4 and/or a CGI score less than 3(borderline mentally ill).

Both NR and R patients were directly interviewed using the DiagnosticInterview for Genetic Studies (DIGS) (Nurnberger et al., Arch. Gen.Psychiatry 51:849-859, 1994), and their medical records werecomprehensively reviewed by a research psychiatrist. Complementaryinformation from the treating physician and nurses in charge was alsoobtained. The severity of the clinical syndrome at the time ofexamination was evaluated by the BPRS, and the diagnosis as meeting thecriteria for NR or R patients was based on all the available data.

The 90 control patients (C) were determined not to have any DSM-IV axisI mental disorders based on DIGS.

All subjects were Caucasian with a majority (52%) of French Canadians.This project was approved by the Research Ethics Board of eachparticipating institution.

Genetic Analysis

Genomic DNA was isolated from peripheral lymphocytes using standardmethods. The target sequence was amplified by PCR using the publishedprocedure of Frosst et al. (Nat. Genet. 10(1):111-113, 1995). PCRproducts were digested with HinfI because the C677T mutation creates aHinfI recognition sequence. The PCR product of 198 bp was digested intofragments of 175 bp and 23 bp if the C677T mutation was present, whilethe wild-type (alanine) allele remained undigested (198 bp). Thedigested PCR products were electrophoresed on 9% polyacrylamide gels andphotographed. Genotyping and scoring were performed by someone blindedto the diagnosis.

Statistical Analysis

The allele frequencies in the groups of schizophrenia subjects andcontrols were compared using a Chi square statistic with one degree offreedom. Because a statistically significant difference between patientsand controls was observed in the frequency of allele V, pair-wisecomparisons between the three groups of subjects (R, NR, and C) weresubsequently conducted. To test for a population bias within thesubjects tested, an analysis between the R, NR, and C patients for whomboth parents were French Canadian and for whom at least one parent didnot have French Canadian ancestry was also performed.

Comparison of Responding, Nonresponding, and Control Groups Based onFrequence of the C677T MTHFR Allele

R and NR schizophrenic patients did not differ significantly accordingto age and gender distributions. Controls were significantly older andincluded more females than the R and NR groups. In keeping with thestudy design, the two groups of schizophrenia patients differedsignificantly according to the severity of psychosis, the percent oftime spent as inpatient since their first contact with a psychiatricinstitution, and their age at first contact with psychiatric carefacilities (Table 8).

Allele V was significantly more frequent in schizophrenic patients,regardless of the quality of their response to neuroleptics andlong-term outcome, compared to controls (χ²=5.44, df=1, p=0.019).However, the most striking finding was observed when patients werestratified according to their quality of response to conventionalneuroleptics and long-term outcome. The group of R patients showed ahighly significant increase in the frequency of allele V (χ²=16.77,df=1, p=0.00004) in contrast to the group of NR patients (χ²=0.053,df=1, p=0.81). The odds ratio associated with carrying at least oneallele V in the group of responder patients was 6.80 with a 95%confidence interval of 5.22 to 8.87.

The same pattern of differences between responders and controls wasobserved when subjects with two French Canadian parents (χ²=8.91, df=1,p=0.002) and those with at least one non-French Canadian parent(χ²=6.71, df=1, p=0.009) were compared separately. The similar resultsobserved in both subpopulations compared to the overall tested Caucasianpopulation reduces the likelihood of a population stratification bias inthe data. The stratification of the data according to gender resulted ina significant difference in males (χ²=12.63, df=1, p=0.0003) and a trendin females (χ²=2.58, df=1, p=0.10).

Both dominant (VV+AV vs. AA) and recessive (VV vs. AV+AA) models oftransmission of the C677T mutation were tested. For both models, thedifferences between neuroleptic responder patients and controls weresignificant for the whole group of patients and for the groupsstratified according to background of parents (Table 9). Finally, anANOVA analysis was performed in which the genotype was the groupingvariable and the age-at-onset was the outcome variable. A tendencytoward a dose effect of allele V was observed for the age-at-onset(F=2.48, df=(2, 94); p=0.088). The average ages at onset±(SD) were 19.16(4.49), 20.91 (5.42) and 22.52 (5.53) for patients with genotypes AA,AV, and VV respectively.

Other Embodiments

While the invention has been described in connection with specificembodiments thereof, it will be understood that it is capable of furthermodifications and this application is intended to cover any variations,uses, or adaptations of the invention following, in general, theprinciples of the invention and including such departures from thepresent disclosure as come within known or customary practice within theart to which the invention pertains and as may be applied to theessential features hereinbefore set forth, and as follows in the scopeof the appended claims.

1. A method of diagnosing a neurological disorder in a subject, saidmethod comprising analyzing the MTHFR nucleic acid in a sample obtainedfrom the subject and determining the presence of a MTHFR mutant alleleat position 677 in the subject that is indicative of the subject havingthe neurological disorder.
 2. The method of claim 1, wherein said mutantallele leads to a decreased level of folate.
 3. The method of claim 1,wherein said mutant allele leads to an increased level of homocysteine.4. The method of claim 1, wherein said mutant allele leads to adecreased level of S-adenosylmethionine or a decreased level ofmethylation reactions.
 5. The method of claim 1, wherein said mutantallele is homozygous.
 6. The method of claim 1, wherein saidneurological disorder is depression.
 7. The method of claim 1, whereinsaid mutant allele is 677 C/T
 8. A method of determining a risk for aneurological disorder or a propensity for the neurological disorder in asubject, said method comprising analyzing the MTHFR nucleic acid in asample obtained from said subject and determining the presence of atleast one MTHFR mutant allele at position 677 in the subject that isindicative of the risk for the neurological disorder or the propensityfor the neurological disorder in the subject.
 9. The method of claim 8,wherein said mutant allele leads to a decreased level of folate.
 10. Themethod of claim 8, wherein said mutant allele leads to an increasedlevel of homocysteine.
 11. The method of claim 8, wherein said mutantallele leads to a decreased level of S-adenosylmethionine or a decreasedlevel of methylation reactions.
 12. The method of claim 8, wherein saidmutant allele is homozygous.
 13. The method of claim 8, wherein saidneurological disorder is depression.
 14. The method of claim 8, whereinsaid mutant allele is 677 C/T